Flow cytometer

ABSTRACT

The disclosed flow cytometer includes a wavelength division multiplexer (WDM). The WDM includes an extended light source providing light that forms an object, a collimating optical element that captures light from the extended light source and projects a magnified image of the object as a first light beam, and a first focusing optical element configured to focus the first light beam to a size smaller than the object of the extended light source to a first semiconductor detector. The disclosed flow cytometer further includes a composite microscope objective to direct light emitted by a particle in a flow channel in a viewing zone of the composite microscope to the extended light source, a fluidic system and a peristaltic pump configured to supply liquid sheath and liquid sample to the flow channel, and a laser diode system to illuminate the particle in the flow channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 61/911,859 entitled “Flow Cytometer,” filedon Dec. 4, 2013. This application is also a continuation-in-part ofInternational Patent Application Serial No. PCT/US2013/043453 entitled“Flow Cytometer,” filed on May 30, 2013, which claims the benefit ofpriority under 35 U.S.C. 119 to U.S. Provisional Patent Application Ser.No. 61/653,245 entitled “Pulseless Peristaltic Pump,” filed on May 30,2012, U.S. Provisional Patent Application Ser. No. 61/653,328 entitled“Composite Microscope Objective with a Dispersion Compensation Plate,”filed on May 30, 2012, U.S. Provisional Patent Application Ser. No.61/715,819 entitled “Wavelength Division Multiplexing for Extended LightSource,” filed on Oct. 18, 2012, U.S. Provisional Patent ApplicationSer. No. 61/715,836 entitled “Diode Laser Based Optical ExcitationSystem,” filed on Oct. 19, 2012, and U.S. Provisional Patent ApplicationSer. No. 61/816,819 entitled “A Simple Fluidic System for SupplyingPulsation Free Liquid to Flow Cell,” filed on Apr. 29, 2013. All of theabove-identified applications are incorporated herein by reference intheir entirety.

BACKGROUND Technical Field

The present disclosure relates generally to the technical field of flowcytometry and, more particularly, to the structure and operation of animproved flow cytometer together with various individual subassembliesincluded therein.

Background

Flow cytometry is a biophysical technique employed in cell counting,sorting, biomarker detection and protein engineering. In flow cytometry,cells suspended in a stream of liquid pass through an electronicdetection apparatus. Flow cytometry allows simultaneous multiparametricanalysis of physical and/or chemical characteristics of up to thousandsof cells per second.

Flow cytometry has various applications including in the fields ofmolecular biology, pathology, immunology, plant biology and marinebiology. Flow cytometry also has broad application in medicine(especially in transplantation, hematology, tumor immunology andchemotherapy, prenatal diagnosis, genetics and sperm sorting for sexpreselection). In marine biology, the autofluorescent properties ofphotosynthetic plankton can be exploited by flow cytometry incharacterizing abundance and community composition. In proteinengineering, flow cytometry is used in conjunction with yeast displayand bacterial display to identify cell surface-displayed proteinvariants with desired properties. A common variation of flow cytometryis physically sorting particles based on their properties therebypurifying a population of interest.

SUMMARY OF DISCLOSURE

The present disclosure provides an improved flow cytometer together withvarious improved components included therein, as well as componentgroups with interacting components.

In certain embodiments, the present disclosure provides a simple andreliable diode laser based optical system capable of delivering afocused laser beam of elliptical cross section with a Gaussian likeintensity distribution along its minor axis and a width along major axisoptimized for flow cytometric applications.

In certain embodiments, the present disclosure provides an imagingquality microscope objective that is easy to manufacture and has longworking distance, large numerical aperture, large field of view andminimal chromatic aberration.

In certain embodiments, the present disclosure provides a simplefluidics system for flow cytometers that is not only reliable, compactand easy to manufacture, but also capable of supporting velocitycritical applications such as in instruments with multiple spatiallyseparated excitation laser beams or in droplet sorters.

In certain embodiments, the present disclosure provides a simple designfor a peristaltic pump providing a pulseless liquid flow.

In certain embodiments, the present disclosure provides a peristalticpump with minimal pulsation.

In certain embodiments, the present disclosure provides a peristalticpump that is simple to manufacture and operate.

In certain embodiments, the present disclosure provides a device capableof collimating a light beam from an extended light source over anextended distance without significantly expanding the beam diameter.

In certain embodiments, the present disclosure provides a WavelengthDivision Multiplexing (WDM) system to separate a light beam intomultiple colored bands. The WDM system may be compatible with low noisesemiconductor detectors. In addition, due to the diversity offluorescent probes, the WDM system may be reconfigurable.

According to an exemplary embodiment, a flow cytometer may include:

1. a Laser Diode (LD) based optical subsystem for impinging a beam oflight upon particles passing through a viewing zone;

2. a composite microscope objective for gathering and imaging lightscattered from or fluoresced by particles passing through the viewingzone;

3. a fluidic subsystem for supplying a liquid sheath flow to the viewingzone;

4. a peristaltic pump for injecting a liquid sample flow carryingparticles that pass together with the liquid sheath flow through theviewing zone;

5. a multimode optical fiber that receives scattered and fluorescedlight from the viewing zone that the composite microscope objectivegathers images; and

6. a wavelength division multiplexer for optically separating lightreceived via the optical fiber into color bands.

According to an exemplary embodiment, the LD based optical subsystem forilluminating particles passing through the flow cytometer's viewing zonemay generally include:

1. a laser diode oriented with its slow axis parallel to the directionof flow;

2. a collimating lens that converts the diverging beam from the LD intoa collimated beam of elliptical shape with its major axis perpendicularto the flow;

3. a focusing lens system that reduces the laser beam at the viewingzone to an optimal width in the direction perpendicular to the flow; and

4. finally a high power cylindrical focusing element placed in theproximity of the viewing zone with its axis perpendicular to thedirection of flow.

The high power cylindrical focusing element may transpose the far fieldprofile of the LD along its slow axis to its Fourier conjugate at theviewing zone along the direction of flow, while maintains the transversebeam profile, such that the laser beam profile at the viewing zone isoptimal for flow cytometric applications.

According to an exemplary embodiment, the composite microscope objectivemay generally include:

1. a concave spherical mirror;

2. a transparent aberration compensation plate with the flow cytometer'sa viewing zone being located between the mirror and the plate. Scatterand fluorescence light emitted from particles in the viewing zone iscollected by the mirror and reflected back toward the compensationplate. Optical aberrations originating from the mirror are significantlyreduced after light passes through the compensation plate. In oneembodiment of the present disclosure, the viewing zone may be locatedinside a flow cell provided by rectangular glass cuvette with a smallrectangular channel through which a particle carrying liquid flows. Theconcave mirror may be made of an optically transparent material, such asglass or optical quality plastics, of plano-convex shape with ahighly-reflective coating on the convex side for internal reflection.The plano-side of the mirror may be either gel-coupled or bonded to oneside surface of the cuvette. The plano-aspheric compensation plate maybe made of a transparent material, such as glass or optical qualityplastics, with the plano side gel-coupled or bonded to the opposite sideof the cuvette. The plano-convex shaped mirror and the asphericcompensation plate may also be formed integrally with the cuvettte. Inyet another embodiment of the present disclosure, the viewing zone maybe in a jet stream with both the concave mirror and the compensationplate being free standing from the viewing zone, and the mirror may be afront surface concave mirror.

According to an exemplary embodiment, the fluidic system may generallyinclude a sheath liquid reservoir from which a liquid pump draws sheathliquid. Sheath liquid then flows from the liquid pump to an inlet of aT-coupling. One outlet arm of the T-coupling connects to a bypass thatreturns a fraction of the pumped sheath liquid back to the sheath liquidreservoir with the returned sheath liquid flowing into air within thesheath liquid reservoir. A second outlet arm of the T-coupling connectsto a sheath route that includes a reservoir capsule followed by aparticle filter and then the flow cell. The sheath liquid exiting theflow cell then goes to the waste tank. The fluidic resistance along thebypass is designed to be lower than the fluidic resistance along thesheath route. Consequently, only a small fraction of the sheath liquidgoes through the flow cell. Note that typical sheath flow rate in flowcytometric applications is a few tens of milliliter per minute. Thebypass therefore permits using higher flow rate liquid pumps that notonly are much less expensive and more reliable, but also operates athigher pulsation frequency which is much easier to attenuate. Since theexit of the bypass route connects to air, it also serves as a largefluidic capacitor for significantly reducing pulsation in sheath liquidflowing along the sheath route. During operation, the inlet portion ofthe filter cartridge is filled with air. Therefore, the filter cartridgealso serves as a fluidic capacitor, for further reducing pulsation inthe sheath liquid at the flow cell to negligible level. Due to the largefluidic resistance at the flow cell, the air trapped near the inlet ofthe filter cartridge becomes compressed. If the liquid pump is turnedoff, the compressed air in filter cartridge being pushed back towardsthe sheath liquid reservoir becomes stored in the reservoir capsulewhose size is chosen to prevent the trapped air from reaching theT-coupling.

According to an exemplary embodiment, the peristaltic pump may generallyinclude a plurality of rollers located at the periphery of a rotor thatmoves the rollers circularly inside a housing's arcuate curved track anda compressible tube that the rollers compress against the track. In oneembodiment of the present disclosure, the track of the peristalticpump's housing may have one recess so the compressible tube isprogressively decompressed to full expansion then compressed to fullclosure every time one of the rollers moves past the recess. Thelocation and shape of the recess maintains the total volume of liquidwithin the compressible tube from the recess to the pump's outletsubstantially invariant. The effect of tube expansion as a roller movespast the pump's outlet is compensated by the tube compression when adifferent other roller immediately upstream of the pump outlet movesinto the recess' compressing section. In another embodiment of thepresent disclosure, the track of the pump housing may contain aplurality of recesses, providing for a plurality of roller upstream ofthe pump outlet to progressively modify the tube compression in multiplesections along the compressible tube. The locations and shapes of theplurality of recesses are designed such that the modification of tubecompression at these sections substantially compensates the effect dueto the tube expansion near the pump outlet. In yet another embodiment ofthe present disclosure, the compressible tube is kept fully closedunderneath the roller except in the inlet and exit sections. A variablespeed motor may be used to drive the pump. When a roller reaches theexit section, the motor's rotation may programmatically speed up tocompensate for the tube's expansion.

According to an exemplary embodiment, a wavelength division multiplexer(“WDM”) may include at least two optical elements. The first opticalelement collimates a beam of light received from an extended lightsource, such as the light from a pinhole or from a multimode opticalfiber. The first optical element magnifies the extended light source,for example, as defined by the pinhole, or the core of the multimodeoptical fiber, to an image having a size similar to the effective crosssection of the first optical element thereby creating a collimated lightbeam between the first optical element and its image. A second opticalelement is positioned near the image, and relays the first opticalelement with unit magnification down the optical path. In this way, thesecond optical element effectively doubles the collimated path length.Additional optical elements in the same 1:1 image relay configurationmay also be included to further extend the collimated optical path. Thecascaded unit-magnification image relay architecture of the presentdisclosure extends the collimated optical path length without large beamexpansion. As a result, WDM techniques well-established in the opticalcommunication industry can be readily adapted for fluorescence lightdetection. In particular, multiple colored bands present in the beam oflight can be separated using dichroic filters located along the opticalpath with the separated light being tightly focused into small spotscompatible with low noise semiconductor photodetectors.

In one embodiment of a WDM, the first optical element is a lens and thesecond element is a concave mirror, although it is apparent to thoseskilled in the art that other types of refractive and/or reflectiveoptical components may also be used to achieve the same design goal. Theoptical path in the WDM of the present disclosure may be folded usingdichroic filters. In one embodiment of the present disclosure, the lightpath may be folded into a zig-zag configuration. To facilitate the flowcytometer's reliable reconfiguration, each dichroic filter may be bondedto a mechanical holder having a reference surface that is opticallyparallel to the filter's reflective surface. As a result, all of theWDM's filters can be accurately positioned along the optical path byreferencing the filter's holder against a common optical flat. Inanother embodiment of the present disclosure, the collimated beampassing through the dichroic filter is further branched out intomultiple colored bands using secondary dichroic filters. It is apparentto those skilled in the art that dichroic filters may be insertedanywhere along the long, narrow and collimated beam path afforded by thepresent disclosure's relay imaging to thereby permit delivering atightly focused beam to photo detectors using a variety of opticalconfigurations, such as the star configuration discussed in U.S. Pat.No. 6,683,314, the branched configuration discussed in U.S. Pat. No.4,727,020 and other types of WDM optical configuration widely practicedin the optical communication industry. Instead of concave mirrors, theWDM may be replaced by curved dichroic filters to further increase thenumber of colored bands selected by the WDM.

According to some exemplary embodiments, an optical system for impingingbeams of light into a viewing zone in which a sample flow carryingobjects and a sheath flow pass through includes a first light source foremitting a first beam of light along a first beam path to illuminateobjects in the viewing zone at a first location, a second light sourcefor emitting a second beam of light along a second beam path toilluminate objects in the viewing zone at a second location, a beamcompressing optical element for reducing widths of the first and secondbeams of light on their major axes to a width less than the width of thesheath flow, and a first chromatic compensation element located on atleast one of the first beam path and the second beam path forcompensating chromatic aberration in the viewing zone such that thefirst location and the second locations are on a common plane parallelto the direction of the sample flow. The wavelength of the second lightsource is different from the wavelength of the first light source. Thechromatic compensation allows compensating the properties of thedifferent paths, resulting e.g. from the different wavelengths, thedifferent path lengths, different locations in the flow path etc. Thisapplies also for multiple compensating elements in different paths, inparticular when using two, three or more wavelengths for illumination.

According to some exemplary embodiments, an optical system includes afirst light source for emitting a first beam of light to illuminateobjects at a first location in a viewing zone, a composite microscopeobjective for imaging light scattered from and fluoresced by the objectsat the first location in the viewing zone at an image plane external tothe composite microscope, and a beam splitter for reflecting ortransmitting scattered and fluoresced light, wherein the light sourceand the image plane are on two sides of the beam splitter. The compositemicroscope includes a concave mirror and an aberration corrector plate.The aberration corrector plate is an aspheric lens that has a first zonewith negative optical power and a second zone with positive opticalpower radially inside the first zone. The viewing zone is positionedbetween the concave mirror and the aberration corrector plate. Thisallows a compact build-up, as the illumination and the detection oflight scattered from and fluoresced by the objects in the viewing zonemay be conducted from the same side of the microscope objective.

According to some exemplary embodiments, an axial light detection systemincludes a concave mirror for reflecting light that propagates from aviewing zone, and a detector for measuring axial light loss produced byan object in the viewing zone by detecting light reflected by theconcave mirror. This allows an effective detection of light loss whichmay serve as a base for better interpretation of the measured values.

According to some exemplary embodiments, a power monitoring system foradjusting power of a light source includes a first light source foremitting a first beam of light, a second light source for emitting asecond beam of light, a first dichroic filter for reflecting the firstbeam of light and passing the second beam of light, a second dichroicfilter for reflecting the second beam of light, a first detector formeasuring residual power of the first and second beams of lightdownstream of the first dichroic filter on a time-division multiplexingbasis, and a control unit coupled with the first detector and the firstand second light sources, wherein the control unit adjusts power of oneor more of the first and second light sources based on measured residualpower of the first and second beams of light by the first detector. Thisallows an effective detection of light power which may serve as a basefor better interpretation of the measured values, as well as aneffective control procedure, in particular when controlling or adaptionrespective light sources.

According to another exemplary embodiment, an optical system includes anobjective adapted for imaging light scattered from and fluoresced by anilluminated object within a viewing zone, an optical transmission memberfor propagating light received from the aspheric lens, a wavelengthdivision multiplexer (WDM) for receiving light propagated by the opticaltransmission member. The objective includes an aspheric lens with afirst zone with negative optical power and a second zone inside thefirst zone with positive optical power, and a concave mirror forreflecting light scattered from and fluoresced by the illuminated objectthrough the aspheric lens, wherein the viewing zone is located betweensaid concave mirror and the aspheric lens. The WDM includes a firstoptical element that produces a beam of light with an image ofsubstantially the same size as the effective size of said first opticalelement, at least one dichroic filter located between said first opticalelement and said image, a second optical element located in one of saidbranches, and an image relay optical element located near the imageproduced by said first optical element in the other branch. The dichroicfilter separates the beam of light into two branches of distinctivecolors. The beam of light in said branch is focused to a spot by saidsecond optical element. The image relay optical element produces animage of said first optical element at substantially unit magnification.This allows an adapted combined operation of the microscope objectiveand the WDM, as well as the optical coupling there between. Inparticular the microscope objective and the WDM as well as the opticalcoupling may be adapted to match to each other with respect towavelength and other parameters.

According to another exemplary embodiment, an optical system includes alight source for emitting a beam of light to illuminate an object in aviewing zone, a concave mirror for receiving and reflecting lightscattered from and fluoresced by the illuminated object, an asphericlens with a first zone with negative optical power and a second zoneinside the first zone with positive optical power, wherein lightreflected by the concave mirror passes through the aspheric lens, andwherein the viewing zone is located between said concave mirror and theaspheric lens, an optical transmission member for receiving andpropagating light from the aspheric lens, and a multiplexer forreceiving light from the optical transmission member and separating thelight into at least two colors. This allows an adapted combinedoperation of the illumination system, the microscope objective and theWDM, as well as the optical coupling there between. In particular theillumination system, the microscope objective and the WDM as well as theoptical coupling may be adapted to match to each other with respect towavelength and other parameters.

According to another exemplary embodiment, an apparatus for imaginglight scattered from and fluoresced by an illuminated object within aviewing zone includes a fluid delivery system for delivering an objectto a viewing zone, a light source for illuminating the object in theviewing zone, a concave mirror located on one side of the viewing zonefor reflecting light scattered from and fluoresced by the illuminatedobject, and an aspheric lens located on another side of the viewing zonefor receiving the light reflected by the concave mirror and forming animage at an image plane, the aspheric lens having a first zone withnegative optical power and a second zone radially inside the first zonewith positive optical power. This allows an adapted combined operationof the fluid delivery system, the illumination system, and themicroscope objective. In particular the fluid delivery system, theillumination system, and the microscope objective may be adapted tomatch to each other with respect to wavelength and other parameters.

According to other exemplary embodiments, an optical method forimpinging beams of light into a viewing zone includes directing a firstbeam of light to illuminate objects in a viewing zone to producescattered and fluoresced light, reflecting the scattered and fluorescedlight using a concave mirror toward an aberration corrector plate,correcting aberrations in the reflected light with the aberrationcorrector plate, wherein the aberration corrector plate has a first zonewith negative optical power a second zone radially inside the first zonewith positive optical power, and reflecting or transmitting thecorrected light using a beam splitter.

According to other exemplary embodiments, an optical method fordetecting light includes reflecting light that propagates from a viewingzone using a concave mirror, and measuring axial light loss produced byan object in the viewing zone by detecting light reflected by theconcave mirror.

According to other exemplary embodiments, a method of gathering andimaging light scattered from or fluoresced by objects in a viewingincludes delivering an object to a viewing zone, illuminating the objectin the viewing zone to produce scattered and fluoresced light,reflecting the scattered and fluoresced light using a concave mirrortoward a transparent aberration corrector plate, and correctingspherical aberrations in the reflected light with the transparentaberration corrector plate, wherein the transparent aberration correctorplate has a first zone with negative optical power and a second zoneradially inside the first zone with positive optical power.

According to other exemplary embodiments, a composite microscopeobjective adapted for imaging light scattered from and fluoresced by anobject present within a viewing zone, comprises a viewing zone, aconcave mirror arrangement, an exit area and an illumination beamforming arrangement, wherein the viewing zone is arranged between theconcave mirror arrangement and the exit area, and wherein the concavemirror is arranged to reflect scattered and fluoresced light impingingfrom an object present in the viewing zone to the exit area, and whereinthe illumination beam forming arrangement is arranged so that anillumination beam entering the illumination beam forming arrangement ispre-definitely formed at the viewing zone. According to other exemplaryembodiments there may be provided an aberration corrector plate, inparticular an aspheric lens in the exit area. This allows an effectivebuild-up of the microscope objective. It should be noted that theaberration corrector plate is not necessary when providing a concavemirror shape allowing a sufficient imaging of the light scattered andfluoresced from an object in the viewing zone. If required an aberrationcorrector plate, in particular an aspheric lens may be arranged in theexit area.

According to other exemplary embodiments a wavelength divisionmultiplexer (WDM) for separating light emitted from a light source intomultiple colored bands comprises an imaging optical arrangement, adichroic filter arrangement, a semiconductor photo detector, and afocusing optical arrangement, wherein the imaging optical arrangementforms a beam of light from the light emitted from a light source andproduces an image of substantially the same size as the effective sizeof said imaging optical arrangement, and wherein the dichroic filterarrangement is located between said imaging optical arrangement and saidimage, and separates the beam of light into a first branch and a secondbranch of distinctive colors, and wherein the semiconductor photodetector is located in the first branch, and wherein the focusingoptical arrangement is located between the dichroic filter arrangementand the semiconductor photo detector so as to focus the beam of lightonto the semiconductor photo detector. Thus, an effective detectionarrangement may be provided, which may be operated with a semiconductordetector. The semiconductor detector may be a semiconductor photodetector. The semiconductor detector may be an avalanche photo diode ora carbon nanotube detector. Thus, a reduced signal to noise ratio can beachieved.

In first aspect of the disclosure, a flow cytometer includes a laserdiode (LD) based optical subsystem for directing a beam of light into aviewing zone of said flow cytometer through which a sample liquidcarrying particles flows, the sample liquid being hydrodynamicallyfocused within the viewing zone by a liquid sheath flow that also flowsthrough the viewing zone, a composite microscope objective for imaginglight scattered from and fluoresced by a particle present within theviewing zone, a fluidic subsystem for supplying the liquid sheath flowto the viewing zone, the liquid sheath flow lacking pulsations, aperistaltic pump for supplying the sample liquid carrying the particles,the sample liquid being hydrodynamically focused within the viewing zoneby the liquid sheath flow, a peristaltic pump for supplying the sampleliquid carrying the particles, the sample liquid being hydrodynamicallyfocused within the viewing zone by the liquid sheath flow, and awavelength division multiplexer (WDM) for separating into multiplecolored bands a beam of light emitted initially from the viewing zoneand imaged by the composite microscope objective into an optical fiberfor transmission to the WDM. The LD based optical subsystem may includea LD for emitting a diverging beam of light from an edge thereof, thediverging beam of light having an elliptically shaped cross-sectionalprofile with both a major axis and a minor axis, a collimating lens forconverting the diverging beam of light emitted from said LD into acollimated elliptical beam of light, wherein the minor axis of saidcollimated elliptical beam of light is oriented parallel to a directionin which particles pass through the viewing zone, a beam compressingoptical el ement for reducing the size of said elliptical beam of lightat the viewing zone whereby a width of said major axis of saidelliptical beam of light oriented perpendicular to the direction inwhich particles pass through the viewing zone is less than a width ofsaid liquid sheath flow, a cylindrical focusing element positionedadjacent to the viewing zone with an axis of said cylindrical focusingelement being oriented perpendicular to the direction in which particlespass through the viewing zone whereby said minor axis of said beam oflight becomes focused at the viewing zone, and the size of said majoraxis of said elliptical beam of light at the viewing zone remainsessentially unchanged. The composite microscope objective may include aconcave mirror upon which scattered and fluoresced light impinges and anaberration corrector plate made of optically transparent material. Theaberration corrector plate is an aspheric lens that has a first zone ofsaid aberration corrector plate having negative optical power outside aneutral zone and a second zone of said aberration corrector plate insidethe neutral zone having positive optical power light. The neutral zoneis the thinnest portion of the aberration corrector plate. Lightreflected from the concave mirror passes through said aberrationcorrector plate. The viewing zone of said flow cytometer is locatedbetween said concave mirror and said aberration corrector plate. Thefluidic subsystem may include a liquid pump for supplying liquid drawnfrom a reservoir and a T-coupling having at least one (1) inlet and two(2) outlets. The inlet of said T-coupling receives liquid from saidliquid pump. A first fraction of the liquid received by the inlet flowsvia a first one of the outlets and via a bypass conduit back to thereservoir. A second fraction of the liquid received by the inlet flowsvia a second one of the outlets and via a particle filter to the viewingzone of said flow cytometer. The peristaltic pump may include a pumphousing having a arcuate curved track formed therein that extendsbetween a pump inlet and a pump outlet, a plurality of rollers that areattached to a rotor, the rollers having a substantially equal angularspacing between each pair of immediately adjacent rollers, the rotorbeing rotatable together with the rollers attached thereto inside saidpump housing, and a compressible tube sandwiched between said rollersand the arcuate curved track of said pump housing. The arcuate curvedtrack includes an exit section and at least one pumping section alongthe arcuate curved track between the pump inlet and the pump outlet. Asa roller rolls through the exit section, said compressible tube adjacentto said roller progressively expands from fully closed at a beginning ofsaid exit section to fully open at the pump outlet where said rollerbreaks contact with said compressible tube. Said compressible tube iscompressed to fully closed by at least one of said rollers. Thewavelength division multiplexer (WDM) may include a collimating opticalelement that magnifies an to produce an image of substantially the samesize as the effective size of said collimating optical element, at leastone dichroic filter located between said collimating optical element andsaid image, said dichroic filter separating the collimated beam of lightinto two (2) branches of distinctive colors, a focusing optical elementlocated in one of said branches, the beam of light in said branch beingfocused to a spot having a diameter of less than 1.0 mm by said focusingoptical element, and an image relay optical element located near theimage produced by said collimating optical element in the other branch,said image relay optical element producing an image of said collimatingoptical element at substantially unit magnification.

In second aspect of the disclosure, said cuvette may have arectangularly-shaped cross-section, and the viewing zone of the flowcytometer is located within a channel having a rectangularly-shapedcross-section that is located within said cuvette.

In third aspect of the disclosure, said cuvette may have atubularly-shaped cross-section, and the viewing zone of the flowcytometer is located within a channel having a circularly-shapedcross-section that is located within said cuvette.

In fourth aspect of the disclosure, the sample liquid and the liquidsheath flow form a jet stream in which the viewing zone of the flowcytometer is located.

In fifth aspect of the disclosure, said cylindrical focusing element isin optical contact with an entrance face of said rectangularly-shapedcuvette.

In sixth aspect of the disclosure, said cylindrical focusing element isseparated from said rectangularly-shaped cuvette.

In seventh aspect of the disclosure, said cylindrical focusing elementis separated from said tubularly-shaped cuvette.

In eighth aspect of the present disclosure, said cylindrical focusingelement is separated from said jet stream.

In ninth aspect of the present disclosure, the flow cytometer furthercomprises a polarization conditioning element through which saidcollimated elliptical beam of light passes.

In tenth aspect of the present disclosure, an optical image of theviewing zone is formed outside the composite microscope objective.

In eleventh aspect of the present disclosure, the viewing zone islocated within a flow channel included in a rectangularly-shaped cuvettemade of optically transparent material.

In twelfth aspect of the present disclosure, said concave mirror is aplano-concave back surface mirror made from an optically transparentmaterial.

In thirteenth aspect of the present disclosure, the plano-surface ofsaid plano-concave back surface mirror is optically coupled to a flatsurface of said cuvette.

In fourteenth aspect of the present disclosure, an optical adhesivematerial accomplishes the optical coupling.

In fifteenth aspect of the present disclosure, an index matching gelaccomplishes the optical coupling.

In sixteenth aspect of the present disclosure, an index matching fluidaccomplishes the optical coupling.

In seventeenth aspect of the present disclosure, optical contact bondingaccomplishes the optical coupling.

In eighteenth aspect of the present disclosure, the plano-concave backsurface mirror formed integrally with said cuvette means.

In nineteenth aspect of the present disclosure, said aberrationcorrector plate is a plano-aspherical lens.

In twentieth aspect of the present disclosure, a plano-surface of saidaberration corrector plate is optically coupled to a flat surface ofsaid cuvette opposite of said plano-concave back surface mirror.

In twenty-first aspect of the present disclosure, an index matching gelaccomplishes the optical coupling.

In twenty-second aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In twenty-third aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In twenty-fourth aspect of the present disclosure, the plano-asphericallens is formed integrally with said cuvette.

In twenty-fifth aspect of the present disclosure, said aberrationcorrector plate is detached from said cuvette.

In twenty-sixth aspect of the present disclosure, the viewing zone isinside a jet stream.

In twenty-seventh aspect of the present disclosure, said concave mirroris a front surface mirror.

In twenty-eighth aspect of the present disclosure, the viewing zone islocated on a surface of a flat, transparent substrate.

In twenty-ninth aspect of the present disclosure, said concave mirror isa plano-concave back surface mirror made from an optically transparentmaterial.

In thirtieth aspect of the present disclosure, the plano-surface of saidplano-concave back surface mirror is optically coupled to said flat,transparent substrate.

In thirty-first aspect of the present disclosure, an optical adhesivematerial accomplishes the optical coupling.

In thirty-second aspect of the present disclosure, an index matching gelaccomplishes the optical coupling.

In thirty-third aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In thirty-fourth aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In thirty-fifth aspect of the present disclosure, said plano-concaveback surface mirror is formed integrally with said flat, transparentsubstrate.

In thirty-sixth aspect of the present disclosure, said aberrationcorrector plate is detached from said flat, transparent substrate.

In thirty-seventh aspect of the present disclosure, the particle filterhas inlet thereto for receiving liquid from the T-coupling, the inlet ofthe particle filter being disposed so that air becomes trapped withinthe particle filter at the inlet thereto.

In thirty-eighth aspect of the present disclosure when said liquid pumpis turned off air cannot enter into the bypass conduit.

In thirty-ninth aspect of the present disclosure, the flow cytometerfurther comprises a small capsule disposed between the second one of theoutlets of said T-coupling and the particle filter for storing airejected from the particle filter when the liquid pump is turned off.

In fortieth aspect of the present disclosure, the flow cytometer furthercomprises a length of tubing disposed between the second one of theoutlets of said T-coupling and the particle filter for storing airejected from the particle filter when the liquid pump is turned off.

In forty-first aspect of the present disclosure, the flow cytometerfurther comprises an adjustable valve located in the bypass conduitbetween the first one of the outlets of the T-coupling and the reservoirfor restricting liquid flow therebetween.

In forty-second aspect of the present disclosure, the flow cytometerfurther comprises an adjustable valve located between the second one ofthe outlets of the T-coupling and the viewing zone for restrictingliquid flow therebetween.

In forty-third aspect of the present disclosure, the throughput of theliquid pump is adjustable.

In forty-fourth aspect of the present disclosure, the arcuate curvedtrack of said pump housing includes at least two (2) pumping sections,the arcuate curved track further including at least one recess sectionlocated between said pumping sections along the arcuate curved track,and said compressible tube at said recess section becoming decompressedto full expansion then compressed to fully closed when one (1) of saidrollers rolls through said recess section.

In forty-fifth aspect of the present disclosure, the peristaltic pumpincludes a plurality of recess sections along said arcuate curved trackupstream of the pump outlet, the angular spacing between the compressionpart of said recess section adjacent to the pump outlet and said exitsection of said arcuate curved track being substantially the same as theangular spacing between each pair of immediately adjacent rollers.

In forty-sixth aspect of the present disclosure, said compression partof said recess section adjacent to the pump outlet has a shapecomplementing a shape of said exit section of said arcuate curved trackto maintain the total fluid volume inside a section of said compressibletube extending from said recess section to the pump outlet substantiallyinvariant when one of said rollers progressively rolls off said exitsection of the arcuate curved track.

In forty-seventh aspect of the present disclosure, the peristaltic pumpincludes a plurality of recess sections respectively interspersedbetween immediately adjacent pairs of a plurality of pumping sections.

In forty-eighth aspect of the present disclosure, both angular spacingbetween adjacent pairs of recess sections, and angular spacing betweensaid exit section of said arcuate curved track and an adjacent recesssection to said exit section are substantially the same as the angularspacing between each pair of immediately adjacent rollers.

In forty-ninth aspect of the present disclosure, shapes of a pluralityof recess sections of said arcuate curved track complement a shape ofsaid exit section of said arcuate curved track to maintain a fluidvolume in sections of said compressible tube at the plurality of recesssections and said exit section substantially invariant when one of saidrollers progressively rolls off said exit section of the arcuate curvedtrack.

In fiftieth aspect of the present disclosure, a speed of said rotor isprogrammably controlled to vary substantially in inverse proportion tothe fluid volume change rate in said compressible tube due to itschanging compression near the exit section of said arcuate curved track.

In fifty-first aspect of the present disclosure, at least one additionaldichroic filter is located between said image relay optical element andthe image produced by said image relay optical element, said dichroicfilter producing two (2) branches of the beam of light havingdistinctive colors.

In fifty-second aspect of the present disclosure, another focusingoptical element is located in one of said branches and focuses the beamof light in the branch into a spot having a diameter of less than 1.0mm.

In fifty-third aspect of the present disclosure, wherein successivecombinations of said image relay optical element, dichroic filter, andfocusing optical element are cascaded to produce additional focusedspots having a diameter of less than 1.0 mm for multiple colored bandsof said beam of light.

In fifty-fourth aspect of the present disclosure, the dichroic filter isassembled using a template that include two (2) optically flat glassplates bonded together in optical contact, and the dichroic filter isbonded to a filter holder using the template such that a coated filtersurface of the dichroic filter is indented and optically parallel to areference surface of the filter holder.

In fifty-fifth aspect of the present disclosure, the reference surfaceof the filter holder rests against an optically flat surface of anreference block included in the WDM thereby providing consistent opticalalignment when installing the dichroic filter into the WDM.

In fifty-sixth aspect of the present disclosure, the LD based opticalsubsystem includes a LD for emitting a diverging beam of light from anedge thereof, the diverging beam of light having an elliptically shapedcross-sectional profile with both a major axis and a minor axis, acollimating lens for converting the diverging beam of light emitted fromsaid LD into a collimated elliptical beam of light, wherein the minoraxis of said collimated elliptical beam of light is oriented parallel toa direction in which particles pass through the viewing zone, a beamcompressing optical element for reducing the size of said ellipticalbeam of light at the viewing zone whereby a width of said major axis ofsaid elliptical beam of light oriented perpendicular to the direction inwhich particles pass through the viewing zone is less than a width ofsaid liquid sheath flow, a cylindrical focusing element positionedadjacent to the viewing zone with an axis of said cylindrical focusingelement being oriented perpendicular to the direction in which particlespass through the viewing zone whereby said minor axis of said beam oflight becomes focused at the viewing zone, and the size of said majoraxis of said elliptical beam of light at the viewing zone remainsessentially unchanged.

In fifty-seventh aspect of the present disclosure, the optical subsystemmay further comprise a cuvette having a rectangularly-shapedcross-section, and the viewing zone may be located within a channelhaving a rectangularly-shaped cross-section that is located within saidcuvette.

In fifty-eighth aspect of the present disclosure, the optical subsystemfurther comprises a cuvette having a tubularly-shaped cross-section, andthe viewing zone is located within a channel having a circularly-shapedcross-section that is located within said cuvette.

In fifty-ninth aspect of the present disclosure, the sample liquid andthe liquid sheath flow form a jet stream in which the viewing zone islocated.

In sixtieth aspect of the present disclosure, cylindrical focusingelement is in optical contact with an entrance face of saidrectangularly-shaped cuvette.

In sixty-first aspect of the present disclosure, said cylindricalfocusing element is separated from said rectangularly-shaped cuvette.

In sixty-second aspect of the present disclosure, said cylindricalfocusing element is separated from said tubularly-shaped cuvette.

In sixty-third aspect of the present disclosure, said cylindricalfocusing element is separated from said jet stream.

In sixty-fourth aspect of the present disclosure, the optical subsystem(50) further comprises a polarization conditioning element through whichsaid collimated elliptical beam of light passes.

In sixty-fifth aspect of the present disclosure, a method for deliveringan elliptically shaped beam of light using a LD based optical subsystem(50), the beam of light having a smooth profile at a focus of a minoraxis thereof that is located at a viewing zone through which a sampleliquid flows, the sample liquid being hydrodynamically focused withinthe viewing zone by a liquid sheath flow that also flows through theviewing zone, the method includes the steps of: providing a LD thatemits a diverging beam of light from an edge thereof, the diverging beamof light having an elliptically shaped cross-sectional profile with botha major axis and a minor axis, impinging the diverging beam of lightemitted by the LD upon a collimating lens for converting the divergingbeam of light emitted therefrom into a collimated elliptical beam oflight wherein the minor axis of said collimated elliptical beam of lightis oriented parallel to a direction in which sample liquid passesthrough the viewing zone, after passing through said collimating lens,impinging the collimated elliptical beam of light upon an beamcompressing optical element for reducing the size of said ellipticalbeam of light at the viewing zone whereby a width of said major axis ofsaid elliptical beam of light oriented perpendicular to the direction inwhich sample liquid passes through the viewing zone becomes less than awidth of said liquid sheath flow, and after passing through said beamcompressing optical element, impinging the beam of light upon acylindrical focusing element positioned adjacent to the viewing zonewith an axis of said cylindrical focusing element being orientedperpendicular to the direction in which sample liquid passes through theviewing zone whereby said minor axis of said beam of light becomesfocused at the viewing zone, and the size of said major axis of saidelliptical beam of light at the viewing zone remains essentiallyunchanged.

In sixty-sixth aspect of the present disclosure, the viewing zone islocated within a channel having a rectangularly-shaped cross-sectionthat is located within a cuvette.

In sixty-seventh aspect of the present disclosure, the viewing zone islocated within a channel having a circularly-shaped cross-section thatis located within a cuvette.

In sixty-eighth aspect of the present disclosure, the viewing zone islocated within a jet stream.

In sixty-ninth aspect of the present disclosure, the method furthercomprises a step of establishing an optical contact between saidcylindrical focusing element and an entrance face of said cuvette.

In seventieth aspect of the present disclosure, the method furthercomprises a step of establishing a spacing between said cylindricalfocusing element and said cuvette.

In seventy-first aspect of the present disclosure, the method furthercomprises a step of establishing a spacing between said cylindricalfocusing element and said cuvette.

In seventy-second aspect of the present disclosure, the method furthercomprise a step of establishing a spacing between said cylindricalfocusing element and said jet stream.

In seventy-third aspect of the present disclosure, the method furthercomprises a step of inserting a polarization conditioning elementbetween the collimating lens and the beam compressing optical elementwhereby the collimated elliptical beam of light passes through thepolarization conditioning element.

In seventy-fourth aspect of the present disclosure, The compositemicroscope objective includes a concave mirror upon which scattered andfluoresced light impinges and an aberration corrector plate made ofoptically transparent material. The aberration corrector plate is anaspheric lens that has a first zone of said aberration corrector platehaving negative optical power outside a neutral zone and a second zoneof said aberration corrector plate inside the neutral zone havingpositive optical power light. The neutral zone is the thinnest portionof the aberration corrector plate. Light reflected from the concavemirror passes through said aberration corrector plate. The viewing zoneof said flow cytometer is located between said concave mirror and saidaberration corrector plate.

In seventy-fifth aspect of the present disclosure, an optical image ofthe viewing zone is formed outside the composite microscope objective.

In seventy-sixth aspect of the present disclosure, the viewing zone islocated within a flow channel included in a rectangularly-shaped cuvettemade of optically transparent material.

In seventy-seventh aspect of the present disclosure, said concave mirroris a plano-concave back surface mirror made from an opticallytransparent material.

In seventy-eighth aspect of the present disclosure, a plano-surface ofsaid plano-concave back surface mirror is optically coupled to a flatsurface of said cuvette.

In seventy-ninth aspect of the present disclosure, an optical adhesivematerial accomplishes the optical coupling.

In eightieth aspect of the present disclosure an index matching gelaccomplishes the optical coupling.

In eighty-first aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In eighty-second aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In eighty-third aspect of the present disclosure, the plano-concave backsurface mirror formed integrally with said cuvette means.

In eighty-fourth aspect of the present disclosure, said aberrationcorrector plate is a plano-aspherical lens.

In eighty-fifth aspect of the present disclosure, a plano-surface ofsaid aberration corrector plate is optically coupled to a flat surfaceof said cuvette opposite of said plano-concave back surface mirror.

In eighty-sixth aspect of the present disclosure, an index matching gelaccomplishes the optical coupling.

In eighty-seventh aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In eighty-eighth aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In eighty-ninth aspect of the present disclosure, the plano-asphericallens is formed integrally with said cuvette.

In ninetieth aspect of the present disclosure said aberration correctorplate is detached from said cuvette.

In ninety-first aspect of the present disclosure, the viewing zone isinside a jet stream.

In ninety-second aspect of the present disclosure, said concave mirroris a front surface mirror.

In ninety-third aspect of the present disclosure, the viewing zone islocated on a surface of a flat, transparent substrate.

In ninety-fourth aspect of the present disclosure, said concave mirroris a plano-concave back surface mirror made from an opticallytransparent material.

In ninety-fifth aspect of the present disclosure, a plano-surface ofsaid plano-concave back surface mirror is optically coupled to saidflat, transparent substrate.

In ninety-sixth aspect of the present disclosure, an optical adhesivematerial accomplishes the optical coupling.

In ninety-seventh aspect of the present disclosure, an index matchinggel accomplishes the optical coupling.

In ninety-eighth aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In ninety-ninth aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In one hundredth aspect of the present disclosure, said plano-concaveback surface mirror is formed integrally with said flat, transparentsubstrate.

In one hundred first aspect of the present disclosure, said aberrationcorrector plate is detached from said flat, transparent substrate.

In one hundred second aspect of the present disclosure, a method forcharacterizing microscopic species using a microscope objective deviceincludes a concave mirror, an aberration corrector plate made ofoptically transparent material, and a viewing zone located in betweensaid concave mirror and said aberration corrector plate. The aberrationcorrector plate is an aspheric lens that has a first zone of saidaberration corrector plate having negative optical power outside aneutral zone and a second zone of said aberration corrector plate insidethe neutral zone having positive optical power light. The neutral zoneis the thinnest portion of the aberration corrector plate.

In one hundred third aspect of the present disclosure, an optical imageof the viewing zone is formed outside the device.

In one hundred fourth aspect of the present disclosure, the viewing zoneis located within a flow channel contained in a rectangularly-shapedcuvette means made of optically transparent material.

In one hundred fifth aspect of the present disclosure, said concavemirror is a plano-concave back surface mirror made from an opticallytransparent material.

In one hundred sixth aspect of the present disclosure, a plano-surfaceof said plano-concave back surface mirror means is optically coupled toa flat surface of said cuvette means.

In one hundred seventh aspect of the present disclosure, an opticaladhesive material accomplishes the optical coupling.

In one hundred eighth aspect of the present disclosure, an indexmatching gel accomplishes the optical coupling.

In one hundred ninth aspect of the present disclosure, an index matchingfluid accomplishes the optical coupling.

In one hundred tenth aspect of the present disclosure, optical contactbonding accomplishes the optical coupling.

In one hundred eleventh aspect of the present disclosure theplano-concave back surface mirror is formed integrally with saidcuvette.

In one hundred twelfth aspect of the present disclosure, said aberrationcorrector plate is a plano-aspherical lens.

In one hundred thirteenth aspect of the present disclosure, aplano-surface of said aberration corrector plate is optically coupled toa flat surface of said cuvette means opposite of said concave mirror.

In one hundred fourteenth aspect of the present disclosure an indexmatching gel accomplishes the optical coupling.

In one hundred fifteenth aspect of the present disclosure, an indexmatching fluid accomplishes the optical coupling.

In one hundred sixteenth aspect of the present disclosure, opticalcontact bonding accomplishes the optical coupling.

In one hundred seventeenth aspect of the present disclosure, theplano-aspherical lens is formed integrally with said cuvette.

In one hundred eighteenth aspect of the present disclosure, saidaberration corrector plate is detached from said cuvette.

In one hundred nineteenth aspect of the present disclosure, the viewingzone is inside a jet stream.

In one hundred twentieth aspect of the present disclosure, said concavemirror is a front surface mirror.

In one hundred twenty-first aspect of the present disclosure, theviewing zone is located on a surface of a flat, transparent substrate.

In one hundred twenty-second aspect of the present disclosure saidconcave mirror is a plano-concave back surface mirror made from anoptically transparent material.

In one hundred twenty-third aspect of the present disclosure, aplano-surface of said plano-concave back surface mirror means isoptically coupled to said flat, transparent substrate.

In one hundred twenty-fourth aspect of the present disclosure, anoptical adhesive material accomplishes the optical coupling.

In one hundred twenty-fifth aspect of the present disclosure, an indexmatching gel accomplishes the optical coupling.

In one hundred twenty-sixth aspect of the present disclosure, an indexmatching fluid accomplishes the optical coupling.

In one hundred twenty-seventh aspect of the present disclosure, opticalcontact bonding accomplishes the optical coupling.

In one hundred twenty-eighth aspect of the present disclosure, saidplano-concave back surface mirror is formed integrally with said flat,transparent substrate.

In one hundred twenty-ninth aspect of the present disclosure, saidaberration corrector plate is detached from said flat, transparentsubstrate.

In one hundred thirtieth aspect of the present disclosure, a fluidicsubsystem for supplying a liquid flow pulsation free to an outlet of thefluidic subsystem includes a liquid pump for supplying liquid drawn froma reservoir and a T-coupling having at least one inlet and two outlets.The inlet of said T-coupling receives liquid from said liquid pump. Afirst fraction of the liquid received by the inlet flows via a first oneof the outlets and via a bypass conduit back to the reservoir. A secondfraction of the liquid received by the inlet flows via a second one ofthe outlets and via a particle filter to the viewing zone of said flowcytometer.

In one hundred thirty-first aspect of the present disclosure, theparticle filter has an inlet thereto for receiving liquid from theT-coupling, the inlet of the particle filter being disposed so that airbecomes trapped within the particle filter at the inlet thereto.

In one hundred thirty-second aspect of the present disclosure, when saidliquid pump is turned off air cannot enter into the bypass conduit.

In one hundred thirty-third aspect of the present disclosure, thefluidic subsystem further comprises a small capsule disposed between thesecond one of the outlets of said T-coupling and the particle filter forstoring air ejected from the particle filter when the liquid pump isturned off.

In one hundred thirty-fourth aspect of the present disclosure, thefluidic subsystem further comprises a length of tubing disposed betweenthe second one of the outlets of said T-coupling and the particle filterfor storing air ejected from the particle filter when the liquid pump isturned off.

In one hundred thirty-fifth aspect of the present disclosure, thefluidic subsystem further comprises an adjustable valve located in thebypass conduit between the first one of the outlets of the T-couplingand the reservoir for restricting liquid flow therebetween.

In one hundred thirty-sixth aspect of the present disclosure, thefluidic subsystem further comprises an adjustable valve located betweenthe second one of the outlets of the T-coupling and the outlet of thefluidic subsystem for restricting liquid flow therebetween.

In one hundred thirty-seventh aspect of the present disclosure, thethroughput of the liquid pump is adjustable.

In one hundred thirty-eighth aspect of the present disclosure, a methodfor supplying a liquid flow pulsation free to an outlet of the fluidicsubsystem includes a liquid pump for supplying liquid drawn from areservoir and a T-coupling having at least one (1) inlet and two (2)outlets. The inlet of said T-coupling receives liquid from said liquidpump. A first fraction of the liquid received by the inlet flows via afirst one of the outlets and via a bypass conduit back to the reservoir.A second fraction of the liquid received by the inlet flows via a secondone of the outlets and via a particle filter to the viewing zone of saidflow cytometer.

In one hundred thirty-ninth aspect of the present disclosure, duringnormal operation certain amount of air is trapped near the inlet portionof said filter cartridge means.

In one hundred fortieth aspect of the present disclosure said reservoirmeans holds sufficient amount of liquid such that when said pump meansis turned off, portion of the tubing between said T-coupling means andsaid reservoir means is still filled with liquid, preventing saidtrapped air from leaking into said bypass means.

In one hundred forty-first aspect of the present disclosure, saidreservoir means is a capsule.

In one hundred forty-second aspect of the present disclosure, saidreservoir means is a piece of tubing.

In one hundred forty-third aspect of the present disclosure anadjustable flow restrictor means is placed in the bypass route.

In one hundred forty-fourth aspect of the present disclosure anadjustable flow restrictor means is placed in the sheath route.

In one hundred forty-fifth aspect of the present disclosure, thethroughput of the sheath pump is adjustable.

In one hundred forty-sixth aspect of the present disclosure, aperistaltic pump includes a pump housing having an arcuate curved trackformed therein that extends between a pump inlet and a pump outlet, aplurality of rollers that are attached to a rotor, the rollers having asubstantially equal angular spacing between each pair of immediatelyadjacent rollers, the rotor being rotatable together with the rollersattached thereto inside said pump housing, and a compressible tubesandwiched between said rollers and the arcuate curved track of saidpump housing. The arcuate curved track includes an exit section and atleast one pumping section along the arcuate curved track between thepump inlet and the pump outlet. As a roller rolls through the exitsection, said compressible tube adjacent to said roller progressivelyexpands from fully closed at a beginning of said exit section to fullyopen at the pump outlet where said roller breaks contact with saidcompressible tube. Said compressible tube is compressed to fully closedby at least one of said rollers.

In one hundred forty-seventh aspect of the present disclosure, thearcuate curved track of said pump housing includes at least two (2)pumping sections, the arcuate curved track further including at leastone recess section located between said pumping sections along thearcuate curved track, and wherein said compressible tube at said recesssection becomes decompressed to full expansion then compressed to fullyclosed when one (1) of said rollers rolls through said recess section.

In one hundred forty-eighth aspect of the present disclosure, theperistaltic pump includes a plurality of recess sections along saidarcuate curved track upstream of the pump outlet, the angular spacingbetween the compression part of said recess section adjacent to the pumpoutlet and said exit section of said arcuate curved track beingsubstantially the same as the angular spacing between each pair ofimmediately adjacent rollers.

In one hundred forty-ninth aspect of the present disclosure, saidcompression part of said recess section adjacent to the pump outlet hasa shape complementing a shape of said exit section of said arcuatecurved track to maintain the total fluid volume inside a section of saidcompressible tube extending from said recess section to the pump outletsubstantially invariant when one of said rollers progressively rolls offsaid exit section of the arcuate curved track.

In one hundred fiftieth aspect of the present disclosure, theperistaltic pump has a plurality of recess sections respectivelyinterspersed between immediately adjacent pairs of a plurality ofpumping sections.

In one hundred fifty-first aspect of the present disclosure, bothangular spacing between adjacent pairs of recess sections, and angularspacing between said exit section of said arcuate curved track and anadjacent recess section to said exit section are substantially the sameas the angular spacing between each pair of immediately adjacent roller.

In one hundred fifty-second aspect of the present disclosure, shapes ofa plurality of recess sections of said arcuate curved track complement ashape of said exit section of said arcuate curved track to maintain afluid volume in sections of said compressible tube at the plurality ofrecess sections and said exit section substantially invariant when oneof said rollers progressively rolls off said exit section of the arcuatecurved track.

In one hundred fifty-third aspect of the present disclosure, a speed ofsaid rotor is programmably controlled to vary substantially in inverseproportion to the fluid volume change rate in said compressible tube dueto its changing compression near the exit section of said arcuate curvedtrack.

In one hundred fifty-fourth aspect of the present disclosure, a methodfor delivering liquid using a peristaltic pump includes a pump housinghaving a arcuate curved track formed therein that extends between a pumpinlet and a pump outlet, a plurality of rollers that are attached to arotor, the rollers having a substantially equal angular spacing betweeneach pair of immediately adjacent rollers, the rotor being rotatabletogether with the rollers attached thereto inside said pump housing, anda compressible tube sandwiched between said rollers and the arcuatecurved track of said pump housing. The arcuate curved track includes anexit section and at least one pumping section along the arcuate curvedtrack between the pump inlet and the pump outlet. As a roller rollsthrough the exit section, said compressible tube adjacent to said rollerprogressively expands from fully closed at a beginning of said exitsection to fully open at the pump outlet where said roller breakscontact with said compressible tube. Said compressible tube iscompressed to fully closed by at least one of said rollers.

In one hundred fifty-fifth aspect of the present disclosure the arcuatecurved track of said pump housing includes at least two (2) pumpingsections, the arcuate curved track further including at least one recesssection located between said pumping sections along the arcuate curvedtrack, and wherein said compressible tube at said recess section becomesdecompressed to full expansion then compressed to fully closed when one(1) of said rollers rolls through said recess section.

In one hundred fifty-sixth aspect of the present disclosure, theperistaltic pump includes a plurality of recess sections along saidarcuate curved track upstream of the pump outlet; The angular spacingbetween the compression part of said recess section adjacent to the pumpoutlet and said exit section of said arcuate curved track beingsubstantially the same as the angular spacing between each pair ofimmediately adjacent rollers.

In one hundred fifty-seventh aspect of the present disclosure, saidcompression part of said recess section adjacent to the pump outlet hasa shape complementing a shape of said exit section of said arcuatecurved track to maintain the total fluid volume inside a section of saidcompressible tube extending from said recess section to the pump outletsubstantially invariant when one of said rollers progressively rolls offsaid exit section of the arcuate curved track.

In one hundred fifty-eighth aspect of the present disclosure, the pumphas a plurality of recess sections respectively interspersed betweenimmediately adjacent pairs of a plurality of pumping sections.

In one hundred fifty-ninth aspect of the present disclosure, bothangular spacing between adjacent pairs of recess sections, and angularspacing between said exit section of said arcuate curved track and anadjacent recess section to said exit section are substantially the sameas the angular spacing between each pair of immediately adjacent roller.

In one hundred sixtieth aspect of the present disclosure, shapes of aplurality of recess sections of said arcuate curved track complement ashape of said exit section of said arcuate curved track to maintain afluid volume in sections of said compressible tube at the plurality ofrecess sections and said exit section substantially invariant when oneof said rollers progressively rolls off said exit section of the arcuatecurved track.

In one hundred sixty-first aspect of the present disclosure, a speed ofsaid rotor of the peristaltic pump is programmably controlled to varysubstantially in inverse proportion to the fluid volume change rate insaid compressible tube due to its changing compression near the exitsection of said arcuate curved track.

In one hundred sixty-second aspect of the present disclosure, thewavelength division multiplexer (WDM) includes a collimating opticalelement that magnifies an to produce an image of substantially the samesize as the effective size of said collimating optical element, at leastone dichroic filter located between said collimating optical element andsaid image, said dichroic filter separating the collimated beam of lightinto two (2) branches of distinctive colors, a focusing optical elementlocated in one of said branches, the beam of light in said branch beingfocused to a spot having a diameter of less than 1.0 mm by said focusingoptical element, and an image relay optical element located near theimage produced by said collimating optical element in the other branch,said image relay optical element producing an image of said collimatingoptical element at substantially unit magnification.

In one hundred sixty-third aspect of the present disclosure, at leastone additional dichroic filter is located between said image relayoptical element and the image produced by said image relay opticalelement, wherein said dichroic filter produces two (2) branches of thebeam of light having distinctive colors.

In one hundred sixty-fourth aspect of the present disclosure, anotherfocusing optical element is located in one of said branches and focusesthe beam of light in the branch into a spot having a diameter of lessthan 1.0 mm.

In one hundred sixty-fifth aspect of the present disclosure, successivecombinations of said image relay optical element, dichroic filter andfocusing optical element are cascaded to produce additional focusedspots having a diameter of less than 1.0 mm for multiple colored bandsof said beam of light.

In one hundred sixty-sixth aspect of the present disclosure, thedichroic filter is assembled using a template that include two (2)optically flat glass plates bonded together in optical contact, andwherein the dichroic filter is bonded to a filter holder using thetemplate such that a coated filter surface of the dichroic filter isindented and optically parallel to a reference surface of the filterholder.

In one hundred sixty-seventh aspect of the present disclosure, thereference surface of the filter holder rests against an optically flatsurface of an reference block included in the WDM thereby providingconsistent optical alignment when installing the dichroic filter intothe WDM.

In one hundred sixty-eighth aspect of the present disclosure, a methodfor separating beam of light into colored bands using a WDM includes acollimating optical element that magnifies an to produce an image ofsubstantially the same size as the effective size of said collimatingoptical element, at least one dichroic filter located between saidcollimating optical element and said image, said dichroic filterseparating the collimated beam of light into two (2) branches ofdistinctive colors, a focusing optical element located in one of saidbranches, the beam of light in said branch being focused to a spothaving a diameter of less than 1.0 mm by said focusing optical element,and an image relay optical element located near the image produced bysaid collimating optical element in the other branch, said image relayoptical element producing an image of said collimating optical elementat substantially unit magnification.

In one hundred sixty-ninth aspect of the present disclosure, at leastone additional dichroic filter may be located between said image relayoptical element and the image produced by said image relay opticalelement, wherein said dichroic filter produces two (2) branches of beamof light having distinctive colors.

In one hundred seventieth aspect of the present disclosure, anotherfocusing optical element is located in one of said branches and focusesthe beam of light in the branch into a spot having a diameter of lessthan 1.0 mm.

In one hundred seventy-first aspect of the present disclosure,successive combinations of said image relay optical element, dichroicfilter and focusing optical element are cascaded to produce additionalfocused spots having a diameter of less than 1.0 mm for multiple coloredbands of said beam of light.

In one hundred seventy-second aspect of the present disclosure, thedichroic filter is assembled using a template that include two (2)optically flat glass plates bonded together in optical contact, andwherein the dichroic filter is bonded to a filter holder using thetemplate such that a coated filter surface of the dichroic filter isindented and optically parallel to a reference surface of the filterholder.

In one hundred seventy-third aspect of the present disclosure, thereference surface of the filter holder rests against an optically flatsurface of an reference block included in the WDM thereby providingconsistent optical alignment when installing the dichroic filter intothe WDM.

In one aspect of the disclosure, a flow cytometer having a wavelengthdivision multiplexer (WDM), which includes an extended light sourceproviding light that forms an object, a collimating optical element thatcaptures light from the extended light source and projects a magnifiedimage of the object as a first light beam, and a first focusing opticalelement configured to focus the first light beam to a size smaller thanthe object of the extended light source to a first semiconductordetector.

In an additional aspect of the disclosure, a flow cytometer includes aviewing zone where a particle in a flow stream is illuminated by light,and a composite microscope objective. The composite microscope objectivefurther includes a concave mirror configured to gather light scatteredfrom or fluoresced by the illuminated particle and to reflect the lightback towards the viewing zone, and an aberration corrector plateconfigured to reduce optical aberrations in the reflected light causedby the concave mirror.

In an additional aspect of the disclosure, a flow cytometer having afluidic system, which includes a liquid pump for supplying liquid drawnfrom a reservoir, and a T-coupling having at least one inlet and twooutlets. The inlet of the T-coupling receives the liquid from the liquidpump. The first fraction of the liquid received by the inlet flows via afirst one of the outlets and via a bypass conduit back to the reservoir.The second fraction of the liquid received by the inlet flows via asecond one of the outlets and via a particle filter to the outlet of thefluidic system.

In an additional aspect of the disclosure, a flow cytometer having aperistaltic pump, which includes a pump housing having an arcuate curvedtrack formed therein that extends between a pump inlet and a pumpoutlet, a plurality of rollers that are attached to a rotor, the rollershaving a substantially equal angular spacing between each pair ofimmediately adjacent rollers, the rotor being rotatable together withthe rollers attached thereto inside the pump housing, a compressibletube sandwiched between the rollers and the arcuate curved track of thepump housing, and a recess section located between the at least twopumping sections. The compressible tube at the recess section is notfully closed. The arcuate curved track further includes an exit sectionand at least two pumping sections along the arcuate curved track betweenthe pump inlet and the pump outlet. As one of the plurality of rollersrolls through the exit section, the compressible tube adjacent to theroller progressively expands from fully closed at a beginning of theexit section to fully open at the pump outlet where the roller breakscontact with the compressible tube. The compressible tube is compressedto fully closed by at least one of the plurality of rollers at the atleast two pumping sections.

In an additional aspect of the disclosure, a flow cytometer having alaser diode (LD) system, which includes a LD for emitting a divergingbeam of light from an edge thereof, the diverging beam of light havingan elliptically shaped cross-sectional profile with both a major axisand a minor axis, a collimating lens for converting the diverging beamof light emitted from the LD into a collimated elliptical beam of light,the minor axis of the collimated elliptical beam of light being orientedparallel to a direction in which particles pass through a viewing zone,a beam compressing optical element for reducing the size of theelliptical beam of light at the viewing zone whereby a width of theelliptical beam of light oriented perpendicular to the direction inwhich the particles pass through the viewing zone is less than a widthof a liquid sheath flow, and a cylindrical focusing element positionedadjacent to the viewing zone with an axis of the cylindrical focusingelement being oriented perpendicular to the direction in which theparticles pass through the viewing zone whereby the minor axis of theelliptical beam of light becomes focused at the viewing zone; and thesize of the major axis of the elliptical beam of light at the viewingzone remains essentially unchanged.

The foregoing has outlined rather broadly the features and technicaladvantages of the present application in order that the detaileddescription that follows may be better understood. Additional featuresand advantages will be described hereinafter which form the subject ofthe claims. It should be appreciated by those skilled in the art thatthe conception and specific aspect disclosed may be readily utilized asa basis for modifying or designing other structures for carrying out thesame purposes of the present application. It should also be realized bythose skilled in the art that such equivalent constructions do notdepart from the spirit and scope of the present application and theappended claims. The novel features which are believed to becharacteristic of aspects, both as to its organization and method ofoperation, together with further objects and advantages will be betterunderstood from the following description when considered in connectionwith the accompanying figures. It is to be expressly understood,however, that each of the figures is provided for the purpose ofillustration and description only and is not intended as a definition ofthe limits of the present claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram schematically illustrating an embodiment of a flowcytometer in accordance with the present disclosure that includes:

a) a LD based optical illumination subsystem;

b) a composite microscope objective upon which light emitted from the LDbased optical illumination subsystem impinges, the composite microscopeobjective having a fluid-passing channel formed therethrough with aparticle illumination viewing zone located inside a cuvette thereof;

c) a fluidic system for supplying a pulsation free flow of sheath liquidto the fluid-passing channel formed through the composite microscopeobjective;

d) a peristaltic pump for introducing a pulsation free flow of sampleliquid that carries cells or particles to be analyzed into the sheathflow of liquid supplied by the fluidic system; and

e) a wavelength division multiplexer (“WDM”) having a zig-zagconfiguration for separating a beam of light into several differentcolored bands, the WDM receiving light via an optical fiber that isscattered from cells or particles as they pass through the compositemicroscope objective's fluid passing channel and are illuminated thereinby light emitted from the LD based optical illumination subsystem.

FIG. 2 is a schematic view depicting a typical high power edge emittingLD that illustrates the fast and slow axes of light emitted therefrom.

FIG. 2A shows a typical far field profile for a laser beam emitted fromthe LD chip depicted in FIG. 2.

FIG. 3A depicts a 3-dimensional view of a conventional LD based opticalillumination subsystem for flow cytometric instruments together with thesystem's flow cell.

FIG. 3B depicts a typical time dependent profile of light scatteringfrom a cell or particle passing through the laser beam depicted in FIG.3A at the focus thereof within the system's flow cell.

FIG. 4A is an elevational view of an alternative prior art LD basedoptical illumination subsystem configuration across the liquid flowingthrough the fluid-passing channel with the beam profile at the focusthereof in the flow cytometer system's viewing zone.

FIG. 4B is a plan view along the liquid flowing through thefluid-passing channel of the alternative prior art LD based opticalillumination subsystem depicted in FIG. 4A.

FIG. 5A is an elevational view across liquid flowing through thefluid-passing channel of the composite microscope objective depicted inFIG. 1, wherein the LD's slow axis oriented transversely to the liquidflow in accordance with one aspect of the present disclosure.

FIG. 5B is a plan view along liquid flowing through the fluid-passingchannel of the composite microscope objective depicted in FIG. 1,wherein the LD's slow axis oriented transversely to the liquid flow inaccordance with one aspect of the present disclosure.

FIG. 5C depicts a typical time dependent profile of light scatteringfrom a cell or particle passing through the fluid passing channel of thecomposite microscope objective depicted in FIG. 1.

FIG. 5D depicts a perspective view of the cylindrical lens of the LDbased optical subsystem coupled with the composite microscope objectivein accordance with one aspect of the present disclosure.

FIG. 5E depicts an enlarged view of the beam profile shown in FIG. 5D.

FIG. 6 is a perspective view of an alternative embodiment of a LD basedoptical illumination subsystem in accordance with one aspect of thepresent disclosure adapted for use in a flow cytometer system in which ajet stream of liquid passes through the viewing zone.

FIG. 6A is an enlarged perspective view of the alternative embodiment ofa LD based optical illumination subsystem of FIG. 6 depicting in greaterdetail the jet stream of liquid passes through the viewing zone.

FIG. 7 is a perspective view of an alternative embodiment of a LD basedoptical illumination subsystem in accordance with one aspect of thepresent disclosure adapted for use in a flow cytometer system whichorients the LD's slow axis parallel to the direction of liquid flowingthrough the fluid-passing channel of the composite microscope objectivedepicted in FIG. 1.

FIG. 8 is a perspective view of a composite microscope objective inaccordance with one aspect of the present disclosure adapted for use inthe flow cytometer system depicted in FIG. 1, the composite microscopeobjective having a fluid-passing channel formed therethrough with theparticle illumination viewing zone located inside a cuvette therein.

FIG. 8A is a perspective view of a combined microscope objective inaccordance with one aspect of the present disclosure.

FIG. 8B illustrates the combined microscope objective depicted in FIG.8A coupled with a flow cell in accordance with one aspect of the presentdisclosure.

FIG. 9A is a cross-sectional elevational view of the compositemicroscope objective taken along the line 9A-9A in FIG. 8 that includesray traces from three (3) spatially separated locations in the viewingzone to an image plane for the objective illustrating scatter andfluorescence emission propagation.

FIGS. 9B1-9B3 are spot diagrams near the image plane depicted in FIG. 9Afor the three (3) spatially separated light emission locations depictedin FIG. 9A.

FIG. 10 is a cross-sectional elevational view similar to that of FIG. 9Afor an alternative embodiment composite microscope objective inaccordance with one aspect of the present disclosure that includes raytraces from three (3) spatially separated locations in a viewing zonetherein to an image plane for the objective illustrating scatter andfluorescence emission propagation.

FIG. 11 is a perspective view of yet another alternative embodiment acomposite microscope objective in accordance with one aspect of thepresent disclosure adapted for use in the flow cytometer system depictedin FIG. 1, the alternative embodiment composite microscope objectivehaving a fluid-passing channel formed therethrough with the particleillumination viewing zone located inside a cuvette therein.

FIG. 12 is a perspective view of an embodiment of a composite microscopeobjective in accordance with one aspect of the present disclosureadapted for use in the flow cytometer system depicted in FIG. 1, adaptedfor use wherein a viewing zone is located inside the jet stream depictedin FIGS. 6 and 6A.

FIG. 13 is a perspective view of an embodiment of a composite microscopeobjective in accordance with one aspect of the present disclosureadapted for use wherein a viewing zone is located on the surface of amicroscope slide.

FIG. 14 is a schematic diagram depicting a fluidic system in accordancewith one aspect of the present disclosure for supplying stable liquidsheath flow to a flow cytometer flow cell that includes:

1. a small capsule located between a sheath liquid pump and the flowcell; and

2. a particle filter located between the small capsule and the flowcell, both the particle filter and the small capsule providing airreservoirs for dampening pump pulsations.

FIG. 15 is a schematic diagram depicting an embodiment of a fluidicsubsystem similar to that illustrated in FIG. 14 that replaces the smallcapsule with a length of tubing for providing an air reservoir.

FIGS. 16A and 16B are histograms that compare measured particle flighttimes at the flow cell when the inlet portion of the particle filter hasair trapped therein (FIG. 16A), and when there is no air within thefluidic subsystem between the sheath liquid pump and the flow cell (FIG.16B).

FIG. 17 is a perspective view of a 3-roller peristaltic pump inaccordance with one aspect of the present disclosure depicting thepump's rollers, tube and surrounding pump housing.

FIGS. 18A through 18D depict simplified views for several states of the3-roller peristaltic pump depicted in FIG. 17 with the rollers indifferent locations.

FIGS. 19 is a detailed longitudinal cross-sectional view of theperistaltic pump's tube being partially compressed by the pump's roller.

FIG. 19A and 19B are detailed cross-sectional views orthogonal to theperistaltic pump's tube's length taken along the lines 19A and 19B inFIG. 19 illustrating the tube's partial compression by the roller.

FIGS. 20A and 20B are schematic diagrams illustrating the pump's rollersand tube viewed along the pump's circular coordinates to depicting thepulseless flow provided by the peristaltic pump.

FIG. 21 is a graph depicting the functional relationship with respect tothe roller position when it rolls off the exit section of thecompressible tube of:

1. the total volume of liquid in the exit half of the pump; as well as

2. the liquid volumes in the pump's:

-   -   a. recess section; and    -   b. exit section.

FIG. 22 is a simplified plan view of a 4-roller peristaltic pump inaccordance with one aspect of the present disclosure.

FIG. 23 is a simplified plan view of a 6-roller peristaltic pump inaccordance with one aspect of the present disclosure.

FIG. 24A is a longitudinal cross-sectional illustration of rollers and acompressible tube for a pulsation minimizing 3-roller peristaltic pumpin accordance with one aspect of the present disclosure having a rotorwith programmable speed.

FIG. 24B is simplified plan view illustrating the pulsation minimizing3-roller peristaltic pump in accordance with one aspect of the presentdisclosure having a rotor with programmable speed.

FIG. 24C is a graph depicting for the pulsation minimizing peristalticpump depicted in FIG. 24B having a programmable speed rotor:

1. negative volume change rate with respect to the roller position;

2. rotor speed; and

3. pump flow rate.

FIG. 25 is a diagram illustrating optical ray tracing for an exemplary 6port wavelength division multiplexer (“WDM”) using a zig-zagconfiguration in accordance with one aspect of the present disclosure.

FIG. 25A illustrates a top view of a light detection assembly of the WDMillustrated in FIG. 25 in accordance with one aspect of the presentdisclosure.

FIG. 25B illustrates a front perspective view of the light detectionassembly of the WDM illustrated in FIGS. 25 and 25A.

FIG. 26 is a diagram illustrating ray tracing of prior art collimatingdevices showing the device's limitation in collimating an extended lightsource.

FIG. 27 is a perspective illustration of an embodiment of a 6 port WDMusing a combination of zig-zag and branched configurations in accordancewith one aspect of the present disclosure.

FIG. 28 is a perspective illustration of another embodiment of a WDMhaving concave dichroic filters in accordance with one aspect of thepresent disclosure.

FIGS. 29A and 29B are perspective illustrations depicting an assemblyprocess for constructing a replaceable dichroic filter assembly for areconfigurable WDM in accordance with the present disclosure.

FIG. 29C is a perspective illustration of a replaceable dichroic filterassembly build in accordance with the illustrations of FIGS. 29A and29B.

FIGS. 30A and 30B are perspective illustrations of a WDM in accordancewith one aspect of the present disclosure depicting installing into theWDM the replaceable dichroic filter assembly depicted in FIG. 29C andits removal therefrom.

FIG. 31 is a diagram schematically illustrating an optical system with asingle light source in accordance with one aspect of the presentdisclosure.

FIG. 32 is a diagram schematically illustrating an optical system withmultiple light sources in accordance with one aspect of the presentdisclosure.

FIG. 33 illustrates an enlarged view of beams of light shown in FIG. 32.

FIG. 34 is a diagram schematically illustrating an optical system withchromatic compensation elements in accordance with one aspect of thepresent disclosure.

FIG. 35 is a diagram schematically illustrating a power monitoringsystem in accordance with one aspect of the present disclosure.

FIG. 36 is a diagram schematically illustrating an optical system inaccordance with one aspect of the present disclosure.

FIG. 37 is a diagram schematically illustrating an axial light lossdetection system in accordance with one aspect of the presentdisclosure.

FIG. 38 is a diagram schematically illustrating an axial light lossdetection system coupled with a second light detection system inaccordance with one aspect of the present disclosure.

DETAILED DESCRIPTION Flow Cytometer

A flow cytometer system may include one or more following components.

1. A flow cell through which a liquid stream, usually called a sheathflow, carries and hydrodynamically aligns cells or particles so thatthey pass single file through the flow cell.

2. A measuring subsystem system coupled to the flow cell that detectscells or particles passing through the flow cell and is usually either:

a. an impedance or conductivity measuring subsystem; or

b. an optical illumination subsystem together with an optical sensingsubsystem.

3. A conversion subsystem for converting the output signal from themeasuring subsystem into computer processable data.

4. A computer for analyzing the data produced by the conversionsubsystem.

The optical illumination subsystem provides a collimated and thenfocused beam of light, usually laser light of a single wavelength, thatimpinges upon the hydrodynamically-focused stream of liquid passingthrough the flow cell. Accordingly, the flow cytometer system may haveone or more light sources that may include:

1. one or more lamps, e.g., mercury or xenon;

2. one or more high-power water-cooled lasers, e.g., argon, krypton ordye laser;

3. one or more low-power air-cooled lasers, e.g., argon (488 nm), HeNe(red-633 nm), HeNe (green) and HeCd (UV); and/or

4. one or more diode lasers (blue, green, red and violet).

The optical sensing subsystem includes one or more detectors aimed wherethe focused liquid stream passes through the light beam. Such detectorsmay include:

1. detectors in line with the light beam (Forward Scatter or F SC);

2. detectors perpendicular to it (Side Scatter or SSC); and

3. fluorescence detectors.

Each suspended particle passing through the beam scatters the light, andfluorescent material present in the particle or attached to the particleexcited by the impinging light emit light at a longer wavelength thanthat of the impinging light.

Detecting and analyzing brightness changes in a combination of scatteredand fluorescent light at each detector (one for each fluorescentemission peak) permits deriving various types of information about thephysical and chemical structure of each individual particle. FSCcorrelates with cell volume. Due to light being scattered off ofinternal components within a cell, SSC depends on the inner complexityof the particle (i.e., shape of the nucleus, the amount and type ofcytoplasmic granules or the membrane roughness). Some flow cytometersomit a fluorescence detector and detect only scattered light. Other flowcytometers form images of each cell's fluorescence, scattered light, andtransmitted light. The flow cytometer system's conversion subsystem,which may include one or more amplifiers which may be either linear orlogarithmic, generally includes one or more Analogue-to-DigitalConverters (“ADCs”) for converting the measuring subsystem's outputsignal into data that is then processed by the computer.

Modern flow cytometers usually include up to four (4) lasers andnumerous fluorescence detectors. Increasing the number of lasers anddetectors permits labeling cells with several different antibodies, andcan more precisely identify a target population by their phenotypicmarkers. Some instruments can even capture digital images of individualcells, allowing for the analysis of a fluorescent signal location withinor on the surface of cells.

FIG. 1 depicts a flow cytometer in accordance with one aspect of thepresent disclosure identified by the general reference number 40. Theflow cytometer 40 may include:

1. a LD based optical subsystem 50;

2. a composite microscope objective 60;

3. a fluidic subsystem 70 for supplying a liquid sheath flow;

4. a peristaltic pump 80 for injecting a liquid sample flow thatcontains particles to be analyzed into the liquid sheath flow suppliedby the fluidic subsystem 70, the liquid sample flow becominghydrodynamically focused by the liquid sheath flow passes through aviewing zone with the composite microscope objective 60 gathering andimaging light scattered and/or fluoresced by particles in the viewingzone;

5. an optical fiber 852 that receives light scattered and/or fluorescedby particles in the viewing zone that the composite microscope objective60 gathers and images;

6. a wavelength division multiplexer 90 (“WDM 90”) for opticallyprocessing scattered and/or fluoresced light received from the opticalfiber 852; and

7. a photodetector system 938 to detect the light processed by the WDM90.

Optical Subsystem 50

In most of the instruments, particles of interest, such as blood cellsor microspheres, are carried by the sheath flow using hydrodynamicfocusing into a viewing zone inside a cuvette or jet stream andilluminated there by a focused laser beam. The technique provides themeans to accurately identify and count particles of interest withoutbeing overwhelmed by background noise occurring outside a registrationtime window (Practical Flow Cytometry, Howard M. Shapiro, Wiley (2003)ISBN 0471411256). To increase detection sensitivity, the cross sectionof the focused laser beam is usually elliptical, with the minor axisalong the direction of flow. In order to maintain the thresholdintegrity, the laser profile must have a smooth or bell shaped profilealong the flow direction. One common method for producing such an beamis to elongate a 5 nearly collimated circular Gaussian beam along thedirection of flow with a beam expander made of either prism orcylindrical lens pair, then focus the beam down with a spherical lens.Since the shape of the beam at the focus is the spatial Fouriertransform of the beam at far field, this produces a Gaussian shapedelliptical spot with minor axis along the flow.

Conventional lasers are expensive, bulky and power hungry. Morerecently, laser diodes (“LD”) have become available. Differing fromconventional lasers, the new generation of LDs is cost effective,compact and power efficient, and shows great promise for new generationof compact biomedical instruments. A LD emits light having an ellipticalcross-section with the ellipse's major axis, frequently called the fastaxis, perpendicular to the LD's junction, and the ellipse's minor axis,frequently called the slow axis, parallel to the LD's junction.Unfortunately, the beam quality of a typical LD, particularly along itsfast axis, leaves much to be desired, preventing its wide acceptance inflow cytometric applications.

In principle, the quality of the LD beam can be significantly improvedby spatial filtering. If a small pinhole or a single mode optical fiberis positioned at the focal point of a lens, such that it only acceptsthe lowest order spatial mode, the beam passing through the pinhole orsingle mode optical fiber will be of nearly perfect Gaussian shape. U.S.Pat. No. 5,788,927 discloses that such a beam can then be collimated andexpanded in the direction of flow through the cytometer, and finallyfocused down to an elliptical shaped Gaussian beam with minor axis alongthe flow direction. Unfortunately, the size of desktop instrumentationlimits the diameter of a pinhole to less than 5 micron. The core size ofa visible wavelength single mode optical fiber also has a similardimension. The challenge to manufacture such a precision spatial filterand maintain its long-term stability not only increases the cost of LDbased laser system, but also reduces its reliability.

More recently, in an effort to reduce the possible side lobes due to theedge effect of limited numerical aperture of collimating lens, U.S. Pat.No. 6,713,019 (“the '019 patent”) discloses rotating the LD by ninetydegrees)(90°) such that its slow axis is parallel to the direction offlow. A beam diffusing section, such as a concave cylindrical lens, isthen introduced to diffuse the collimated beam in the directionperpendicular to the flow, followed by a beam spot forming section, suchas a spherical focusing lens, to form an elliptical spot within thecytometer's particle viewing zone. As described in detail in the '019patent, the laser beam after the spot forming section is extremelyastigmatic. In particular, the width of the beam at the viewing zone inthe direction perpendicular to the flow is comparable or even wider thanthe width of the flow channel. This not only reduces the amount of laserenergy impinging upon the particle and consequently the signalintensity, but also increases undesired background scattering from theliquid-flow cell interface. Instead of rotating the LD, U.S. Pat. Nos.7,385,682 and 7,561,267 disclose using a large numerical apertureaspheric lens for LD collimation. Such a design, however, cannot correctthe fringe effect inherent in the LD's beam profile. Consequently, therepresently exists a need for a simple LD based optical system for use inflow cytometers that can reliably produce a focused elliptical beam withnear Gaussian shape along its minor axis and a width along major axis.

In accordance with one aspect of the present disclosure, the opticalsubsystem 50 may include a LD 501 that, as depicted in greater detail inFIG. 2, emits a diverging beam of light from an edge thereof. As moregraphically depicted in FIGS. 2 and 2A, the diverging beam of light hasan elliptically shaped cross-sectional profile with both a major axis,a.k.a. the fast axis, and a minor axis, a.k.a. the slow axis. Thediverging beam of light emitted from the LD 501 may impinge upon acollimating lens 502 which converts the diverging beam of light emittedby LD 501 into a collimated beam of light having an ellipticalcross-section. Although not essential, the optical subsystem 50 may alsoinclude an optional mirror 503 positioned to direct the collimatedelliptical beam of light towards the composite microscope objective 60.A plano-convex lens 504, positioned near the composite microscopeobjective 60, may reduce the major axis of the elliptically shaped beamof light that is oriented perpendicular to the direction in which theliquid sample and the surrounding liquid sheath flow through the viewingzone within the composite microscope objective 60. At the viewing zone,the width of the elliptically shaped beam of light:

1. perpendicular to the direction in which the liquid sample flow passesthrough the viewing zone may be slightly less than the width of theliquid sheath flow; while

2. still sufficiently wide so particles in the sample flow pass througha nearly flat portion of the elliptically shaped beam of light at thebeam's maximum intensity.

In accordance with one aspect of the present disclosure, it is apparentto those skilled in the art that the plano-convex lens 504 may bereplaced by other types of optical elements such as an achromaticdoublet lens or combination of spherical lenses, cylindrical lenses,and/or prism pairs. Alternatively, the mirror 503 and the lens 504 mayalso be replaced by a concave mirror. For polarization sensitiveapplications of the flow cytometer 40, an optional polarizationconditioning element, such as a half-wave plate, may also be placed inthe collimated section of the beam of light extending from thecollimating lens 502 to the lens 504. Finally, before passing throughthe viewing zone the beam of light may pass through a high powercylindrical lens 505, positioned adjacent to the viewing zone. Asdepicted in FIG. 1, the axis of the cylindrical lens 505 is orientedperpendicular to the direction in which the liquid sample flow passesthrough the viewing zone, and the focal length of the cylindrical lens505 produces a tight focusing of the beam of light's minor axis at theviewing zone.

An advantage of the optical subsystem 50 in comparison with conventionalLD based optical subsystem may be discerned more clearly in FIGS. 2 and2A. Most commercially available laser diodes suitable for use in a flowcytometer emit a beam of light from an edge thereof. As depicted in FIG.2, a gain section 509 of such a LD chip 510 is highly confined in thetransverse direction indicated by an arrow 511. Consequently, to achievehigh output power LD manufacturers often sacrifice beam quality,particularly along the transverse or fast axis direction that isoriented parallel to the arrow 511. FIG. 2A shows this characteristic oflight emitted from a LD wherein multiple fringes 512 due to gainconfinement are clearly visible at the far field in the minor axisdirection of the emitted beam of light. It should be noted that thefringes 512 appearing in the illustration of FIG. 2A contain only aminor amount of total energy in the beam of light, and therefore havelittle impact on the conventional M-square characterization of thecorresponding beam profile. However, as discussed in greater detailbelow, the fringes 512 do have a detrimental effect on the performanceof conventional flow cytometers. Alternatively, gain confinement alongthe slow axis direction of an edge emitting LD that is orientedperpendicular to the arrow 511 is much more relaxed. Consequently, asshown in FIG. 2A, the far field beam profile is much smoother along theslow axis of the LD's beam of light.

FIG. 3A depicts a conventional LD based optical subsystem for a flowcytometer. Those elements depicted in FIG. 3A that are common to theoptical subsystem 50 illustrated in FIG. 1 carry the same referencenumeral distinguished by a prime (′) designation. As depicted in FIG.3A, the conventional optical subsystem orients the fast axis of the LD501 parallel to the direction in which the liquid sample flow passesthrough the viewing zone. In its most simplified configuration, theelliptical beam profile of the LD 501′ is directly transposed by thespherical focusing lens 504′ into the viewing zone. In an attempt toachieve optimal aspect ratio for the focused beam of light, variousdifferent conventional LD based optical subsystems have also includedbeam shaping optical elements in addition to those depicted in FIG. 3A.

The detrimental effect of fringes 512 along the fast axis of the LD 501′for conventional optical subsystem configurations clearly appears in thelight scattering time profile depicted in FIG. 3B. Since scattering orfluorescence intensity is directly proportional to the local laser powerimpinging upon a particle, any fine structure in the beam of light'sprofile along the direction in which the liquid sample flow passesthrough the viewing zone will appear in the time profile of the signalproduced by the flow cytometer. Such structures in the time profile areindistinguishable from signals generated by small particles, and willtherefore cause the flow cytometer to trigger falsely and misidentifyparticles. In addition, the fringes 512 will also lead to uncertainty inthe measurement of other cytometric parameters, such as in the area andwidth of the pulse depicted in FIG. 3B.

FIGS. 4A and 4B depict a yet another prior art optical subsystem for LDbased flow cytometric applications disclosed in the '019 patentidentified previously. Those elements depicted in FIGS. 4A and 4B thatare common to the optical subsystem 50 illustrated either in FIG. 1 or3A carry the same reference numeral distinguished by a double prime (″)designation. As depicted in FIGS. 4A and 4B, by orienting the slow axisof the LD 501″ parallel to the direction in which the liquid sample flowpasses through the viewing zone the optical subsystem depicted in FIGS.4A & 4B effectively overcomes the problem caused by the fringes 512 asdescribed above. Unfortunately, the beam-diffusing element 513″ placedbefore the spherical focusing lens 504″ in FIGS. 4A and 4B to diffusethe beam of light perpendicular to the direction in which the liquidsample flow passes through the viewing zone produces a highly astigmaticbeam of light near the viewing zone. Specifically, focusing thisastigmatic beam of light at the viewing zone in the direction in whichthe liquid sample flow passes through the viewing zone increases thewidth of the beam of light perpendicular to the direction in which theliquid sample flow passes through the viewing zone so the beam's widthbecome similar to or even wider than the sheath flow. Consequently, theoptical subsystem depicted in FIGS. 4A and 4B not only diminishes theamount of light energy impinging upon particles flowing through theviewing zone, the optical subsystem also increases undesirablescattering of light from the interface between the liquid sheath flowand adjacent parts of the composite microscope objective 60.

FIG. 5 highlights the main differences between the optical subsystemdisclosed in the '019 patent and the optical subsystem 50 depicted inFIG. 1. Instead of placing an out-of-plane beam-diffusing element 513″before the spherical beam focusing lens 504 as shown in FIG. 4, the highpower cylindrical lens 505, depicted in FIGS. 5A and 5B as a cylindricalplano-convex lens, may be placed along the beam of light after thespherical beam focusing lens 504 and may be juxtaposed with thecomposite microscope objective 60. As shown in FIGS. 5A and 5B, thecylindrical lens 505 may focus the minor axis of the beam of light inthe viewing zone while leaving the major axis of the beam of lightessentially unchanged. Consequently, the optical subsystem 50 depictedin FIGS. 1, 5A and 5B may establish a beam of light profile at theviewing zone which is elliptical with:

1. a tightly focused minor axis that spans across the combined liquidsample and sheath flows; and

2. a smooth minor axis profile in the direction of the combined liquidsample and sheath flows that is the Fourier conjugate of the far fieldbeam profile along the slow axis of LD 501.

Meanwhile, as shown in FIG. 5B, the out-of-plane beam width may beunaffected by the cylindrical lens 505. FIG. 5C shows a measured timeprofile of light scattered from a micro particle using the opticalsubsystem 50 depicted in FIGS. 1, 5A and 5B. The LD 501 used in makingthe measurement presented in FIG. 5C is the same as that used ingenerating the measured time profile of light scattered from a microparticle appearing in FIG. 3B. As shown in FIG. 5C, the side lobescaused by the fringes 512 along the fast axis of the LD 501 no longerhave any material effect on performance of the flow cytometer 40.

FIG. 5D illustrates a perspective view of the cylindrical lens 505 ofthe LD based optical subsystem 50 coupled with the composite microscopeobjective 60 in accordance with some embodiments of the presentdisclosure. A beam of light may pass through the cylindrical lens 505and a cuvette 603 of the microscope objective 60 substantially along thez axis and establish a beam profile 524 on a x-y plane at the viewingzone inside a flow channel 604 of the composite microscope objective 60.

FIG. 5E illustrates an enlarged view of the beam profile 524 shown inFIG. 5D in accordance with some embodiments of the present disclosure.FIG. 5E shows that the minor axis of the beam of light may be along they axis and substantially parallel to the direction of liquid sample andsheath flows and the major axis of the beam of light may be along the xaxis and substantially perpendicular to the direction of liquid sampleand sheath flows.

FIG. 6 depicts yet another alternative diode laser based opticalsubsystem in accordance with some embodiments of the present disclosureadapted for use in a flow cytometer. Those elements depicted in FIGS. 6and 6A that are common to the optical subsystem 50 illustrated in FIGS.1, 5A and 5B carry the same reference numeral distinguished by a tripleprime (′″) designation. The optical subsystem 50′″ depicted in FIGS. 6Aand 6B is almost identical to that shown in FIGS. 1, 5A and 5B exceptthat the viewing zone occurs without a composite microscope objective 60because it occurs in a free-flowing jet stream 519 that includes boththe sample and sheath flows that is emitted from a nozzle 518.Consequently, for the configuration of the optical subsystem 50′″depicted in FIGS. 6A and 6B, the high power cylindrical lens 505 isdetached from the viewing zone that is located within the jet stream519.

In the exemplary embodiments of the present disclosure depicted in FIGS.1, 5A, 5B, 6A and 6B, the minor axis, i.e. the slow axis, of the LD 501is substantially oriented perpendicular to the direction in which theliquid sample flow passes through the viewing zone. However, it will beapparent to those skilled in the art that using an alternative opticalconfiguration the major axis, i.e. the fast axis, of the LD 501 may bereoriented to be perpendicular to the direction in which the liquidsample flow passes through the viewing zone. FIG. 7 depicts one exampleof such an alternative configuration of optical elements.

Those elements depicted in FIG. 7 that are common to the opticalsubsystem 50 illustrated in FIGS. 1, 5A, 5B, 6A and 6B carry the samereference numeral distinguished by a quadruple prime (″″) designation.As shown, the slow axis of the LD 501″″ is oriented in the z-direction.The beam of light emitted from the LD 501″″ is then rotated to thein-plane y-direction by a pair of ninety degrees)(90°)reflection mirrors523 a and 523 b. In the illustration of FIG. 7, a normal to the firstelliptically-shaped light beam reorienting mirror 523 a is oriented inthe x-y plane at forty-five degrees)(45°) to the x-axis, and a normal tothe second elliptically-shaped light beam reorienting mirror 523 b isoriented in the y-z plane at forty-five degrees)(45°) to the z-axis.

Composite Microscope Objective 60

Modern flow cytometers include a spatial filter, usually either amechanical pinhole or a large core optical fiber, located at an imagelocation of an objective lens to prevent undesired background light fromentering the cytometer's detector(s). Because particles remain in thecytometer's viewing zone for a few microseconds, microscope objectiveswith large numerical aperture must be used to maximize light collectionefficiency. To support multiple spatially separated excitation laserbeams in flow cytometers, as disclosed in U.S. Pat. No. 4,727,020, it isalso desirable to use an objective with large field of view. In order toachieve these goals, U.S. Pat. Nos. 6,5100,07 and 7,110,192 disclose anobjective design using a modified apochromat with a gel-coupled or epoxybonded near hemisphere lens as the optical element closest to the samplethat is followed by multiple meniscus lenses. While such microscopeobjectives provide both a satisfactory numerical aperture and field ofview, they significantly sacrificed image quality thereby:

1. limiting effective use of the spatial filter; and

2. exhibiting poor background light discrimination.

Further, such refractive microscope objectives are bulky, expensive tomanufacture and often exhibit severe chromatic aberration. To overcomethese limitations, Published Patent Cooperation Treaty (“PCT”) PatentApplication No. WO 01/27590 discloses an alternative objective designbased on a spherical concave mirror. The design offers large numericalaperture and good image quality along the optical axis. However, due toits poor off-axis characteristics, such a design is unsuitable for flowcytometers having multiple, spatially separated laser beams.

FIG. 8 depicts one embodiment in accordance with the present disclosurefor the composite microscope objective 60 depicted in FIGS. 1, 5A, 5Band 7. As depicted in FIG. 8, the composite microscope objective 60 mayimage a viewing zone that is located inside a prismatically-shaped glasscuvette 603 within a small flow channel 604, that may have a rectangularcross-sectional shape, through which passes the particle carried bycombined liquid sample and sheath flows. A plano-concave back-surfacemirror 601 included in the composite microscope objective 60 may be madeof an optically transparent material that may have a refractive indexsimilar to that of the glass cuvette 603, such as glass or opticalquality plastics. To minimize optical loss, the back-surface mirror 601may include a flat front surface that is optically coupled to anabutting flat surface of the prismatically-shaped cuvette 603. Opticalcoupling of the back-surface mirror 601 to the cuvette 603 may employ anindex-matching gel, optical adhesive or direct optical bonding.Alternatively, the back-surface mirror 601 may also be formed integrallywith the cuvette 603.

The composite microscope objective 60 may also include a plano-asphericcorrector plate 602 that is also made of an optically transparentmaterial that may have a refractive index similar to that of the glasscuvette 603, such as glass or optical quality plastics. To reduceoptical loss, a flat surface of the corrector plate 602 may be opticallycoupled to an abutting flat surface of the prismatically-shaped cuvette603 on a face thereof that is diametrically opposite to the back-surfacemirror 601. Optical coupling of the corrector plate 602 to the cuvette603 may employ an index-matching gel, optical adhesive or direct opticalbonding. The aspheric surface of the corrector plate 602 furthest fromthe corrector plate 602 may carry an anti-reflective coating to reduceoptical transmission loss, although such a coating is not a mandatoryrequirement for a composite microscope objective 60 in accordance withsome embodiments of the present disclosure. The shape of the asphericsurface of the corrector plate 602 is similar to that in a classicalSchmidt camera, (Schmidt, B., Mitt. Hamburg Sternwart 7 (36) 1932). Asknown by those skilled in the art, the corrector plate of a Schmidtcamera includes a circularly shaped neutral zone where the correctorplate does not deviate rays of light passing through the plate. For usein the composite microscope objective 60, outside of the neutral zone ofthe corrector plate 602, where the plate thickness is thinnest, thecorrector plate 602 may have negative optical power while inside theneutral zone the corrector plate 602 may have positive optical power.The exact shape of the aspheric corrector plate 602 may be readilyobtained using any commercially available optical ray tracing tool byany person having ordinary skill in the art. Note that in the flowcytometer 40, the beam of light generated by the optical subsystem 50depicted in FIGS. 1, 5A, 5B and 7 enters the cuvette 603 perpendicularlyto the flow channel 604 through one (1) of the two (2) faces of thecuvette 603 that do not abut the back-surface mirror 601 or correctorplate 602.

Combined Microscope Objective 65

FIG. 8A illustrates a perspective view of a combined microscopeobjective 65 in accordance with some embodiments of the presentdisclosure. The combined microscope objective 65 may include thecomposite microscope objective 60, as illustrated in FIG. 8 and thecylindrical lens 505. The cylindrical lens 505 may direct a beam oflight to the viewing zone in the flow channel 604 to illuminateparticles in the sample flow. After particles are illuminated in theviewing zone, the composite microscope objective 60 then may collectimaging light scattered from and fluoresced by particles within the viewzone.

FIG. 8B illustrates a perspective view of the combined microscopeobjective 65 coupled with a flow cell 619 in according with someembodiments of the present disclosure. Liquid sample 623 may be pumpedup from a sample tube 621 into a flow section 620 of the flow cell 619by a pump 624. The pump 624 may be the peristaltic pump 80, asillustrated in FIG. 17. Liquid sheath 622 may be also pumped into theflow section 620 of the flow cell 619. The pump for pumping the liquidsheath 622 into the flow cell 619 may be a part of the fluidic system70, as illustrated in FIG. 14 or 15. The liquid sample 623 may becombined with the liquid sheath 622 in the flow section 620 of the flowcell 619 and then hydro-dynamically focused within the viewing zoneinside the flow channel 604 of the combined microscope objective 65. Thecombined microscope objective 65 or the composite microscope objective60 may be positioned on the flow cell 619. Persons skilled in the artmay also refer to a combination of the microscope objective 65 or thecomposite microscope objective 60 and the flow cell 619 as a flow cell.The cross-sectional area of the flow section 620 at the top of the flowcell 619 may be smaller than the cross-sectional area of the flowsection 620 at the bottom of the flow cell 619 to facilitatehydrodynamic focusing of the liquid sample 623 in the viewing zone. Itshould be noted that the various aspects of the present disclosure arenot limited to specific direction of liquid sheath or sample flow andspecific shape of the flow cell or the microscope objective.

FIG. 9A depicts the result of ray tracing for the embodiment ofcomposite microscope objective 60 illustrated in FIG. 8. As depicted inFIG. 9A, scatter and fluorescence emissions from three (3) spatiallyseparated locations in the flow channel 604 near the center of thecuvette 603 may:

1. initially propagate toward back-surface mirror 601 and pass firstthrough the cuvette 603 to be internally reflected by the back-surfacemirror 601;

2. then pass through the cuvette 603;

3. subsequently pass through the aspheric corrector plate 602; and

4. finally forms three (3) distinct images near an image plane 605.

Note that rays traversing the composite microscope objective 60 depictedin FIG. 9A are nearly optically-uniform and that light emitted near thecenter of the cuvette 603 traverses the corrector plate 602 at nearnormal incidence. Consequently, the composite microscope objective 60introduces very little chromatic dispersion in light emitted near thecenter of the cuvette 603.

Further, it is well known in the astrophysics community that Schmidtcamera offers the unparalleled combination of a fast focal ratio and alarge field of view with near diffraction limited optical performance.The principal drawback in a conventional Schmidt camera is that theimage surface lies inside the instrument. For the composite microscopeobjective 60, light near the center of the cuvette 603 propagatesopposite to that of a conventional Schmidt camera and therefore theimage surface lies outside the composite microscope objective 60.Consequently, the present disclosure takes full advantage of the opticalperformance of the Schmidt camera without experiencing its limitation.FIGS. 9B1 through 9B3 depict spot diagrams near the image plane 605 forthree (3) emission locations, 606, 607, 608 in viewing zone within theflow channel 604 that may be separated 150 micron from each other. Thediameters of all images depicted in FIGS. 9B1 through 9B3 may be lessthan 35 microns.

Light emitted from the viewing zone within the flow channel 604 of thecomposite microscope objective 60 depicted in FIGS. 8 and 9A thattraverses the aspheric corrector plate 602 may suffer from a smallamount of chromatic aberration. FIG. 10 depicts an alternativeembodiment for the composite microscope objective 60 depicted in FIGS.1, 5A, 5B and 7 in accordance with some embodiments of the presentdisclosure. Those elements depicted in FIG. 10 that are common to thecomposite microscope objective 60 illustrated in FIGS. 8 and 9A carrythe same reference numeral distinguished by a prime (′) designation. Theshapes of the back-surface mirror 601′ and the aberration correctorplate 602′ depicted in FIG. 10 are modified slightly to producecollimated afocal images of the emission locations near the viewing zonewithin the flow channel 604′. In FIG. 10 the composite microscopeobjective 60′ may include a chromatic compensating doublet lens 609inserted between the corrector plate 602′ and the image plane 605′. Inaddition to focusing the light emitted from the corrector plate 602′onto the image plane 605′, the doublet lens 609 may also serve tofurther reduce the residual chromatic aberration introduced by theaspheric corrector plate 602′.

It is not essential that the flat surface of the corrector plate 602 tobe optically coupled to the cuvette 603. FIG. 11 depicts an alternativeembodiment of the composite microscope objective 60 in accordance withsome embodiments of the present disclosure. Those elements depicted inFIG. 11 that are common to the composite microscope objective 60illustrated in FIGS. 8 and 9A carry the same reference numeraldistinguished by a double prime (″) designation. FIG. 11 depicts theaberration corrector plate 602″ optically decoupled from the cuvette603″. Although not essential for operation of the composite microscopeobjective 60″, to improve the light transmission efficiency bothsurfaces of the corrector plate 602″ and the exposed flat surface of thecuvette 603″ may carry an anti-reflectively coating. It is understoodthat the corrector plate 602″ shown in FIG. 11 may be held in fixedrelationship to the combined back-surface mirror 601 and cuvette 603 bya mechanical support not depicted in FIG. 11. Similar to the compositemicroscope objective 60 and 60′ depicted respectively in FIGS. 9A and10, the composite microscope objective 60″ with detached corrector plate602″ may be configured to provide either finite focal length image, oran afocal system which in turn is focused to a finite distance imageplane by an additional chromatic compensating doublet lens 609.

FIG. 12 depicts yet another alternative embodiment of the compositemicroscope objective 60. Those elements depicted in FIG. 12 that arecommon to the composite microscope objective 60 illustrated in FIGS. 8,9A and 11 carry the same reference numeral distinguished by a tripleprime (′″) designation. The composite microscope objective 60′″ depictedin FIG. 12 is adapted for collecting scatter and fluorescence emissionsfrom cells or other microscopic particles carried in the jet stream 519emitted by the nozzle 518. The composite microscope objective 60′″ mayinclude a concave, spherically shaped, front surface mirror 610 and anaberration corrector plate 612. The front surface mirror 610 may be madeof glass or other types of hard material with a highly reflectivecoating on the concave surface 611 or made of metal with polishedconcave surface 611. Similar to the corrector plate 602, theplano-aspheric corrector plate 612 may be made of a thin piece oftransparent material, such as glass or optical quality plastics. Theaspheric surface may be formed on either side of the corrector plate612. Both surfaces of the corrector plate 612 may be coated with ananti-reflective coating to reduce optical transmission loss, althoughsuch a coating is not a mandatory requirement for a corrector plate 612in accordance with some embodiments of the present disclosure. It isunderstood that the front surface mirror 610 and the corrector plate 612may be held in fixed relationship to each other by a mechanical supportnot depicted in FIG. 12. Scatter and fluorescence light emitted fromcells or other types of microscopic particles in the viewing zone insidethe jet stream 519 may be reflected by the concave surface 611 of thefront surface mirror 610. The aberration due to reflection from theconcave surface 611 may be corrected by the corrector plate 612 afterlight traverses through the corrector plate 612. It is understood bythose having skill in the art that the composite microscope objective60′″ may be configured to provide either a finite focused image similarto that depicted in FIG. 9A, or a collimated afocal image which isfocused at finite distance from the composite microscope objective 60′″by a chromatic aberration correction doublet similar to the doublet lens609 depicted in FIG. 10.

FIG. 13 depicts an adaptation of the composite microscope objective 60for imaging specimens fixed to the surface of a transparent substratesuch as a glass slide. Those elements depicted in FIG. 13 that arecommon to the composite microscope objective 60 illustrated in FIGS. 8,9A and 11 carry the same reference numeral distinguished by a quadrupleprime (″″) designation. The composite microscope objective 60″″ depictedin FIG. 13 may include two (2) optical elements, one a plano-concaveback surface mirror 617 made of a transparent material, such as glass oroptical quality plastics, and an aberration corrector plate 618. Asdepicted in FIG. 13, the specimen to be imaged may be fixed to a frontsurface 615 of a transparent, usually glass slide 616. The slide 616 maybe optically coupled, for example, using a thin layer of index matchingfluid, to the flat surface of the back surface mirror 617. Scatter andfluorescence light emitted by the specimen may:

1. initially propagate through the slide 616 and the back surface mirror617;

2. be internally reflected by the back surface mirror 617 back throughthe slide 616;

3. then pass through the corrector plate 618; and

4. finally form an image at an image plane that is located beyond thecorrector plate 618.

Fluidic Subsystem 70

The performance of a flow cytometer depends critically on a stableliquid sheath flow. In particular, flow cytometers that have multiplespatially separated excitation laser beams or perform droplet sortingrely on a constant velocity of the liquid sheath flow for timingsynchronization. As disclosed in U.S. Pat. No. 5,245,318, conventionalflow cytometers provide a stable liquid sheath flow by using an airtightfluidic system that either:

1. applies constant air pressure in a sheath liquid reservoir to pushthe fluid through the flow cell; or

2. by sucking the fluid from the sheath liquid reservoir through theflow cell using a vacuum pump.

These systems are bulky, expensive to manufacture, and prone to failure.More recently, U.S. Pat. No. 8,187,888 discloses including a sheathliquid subsystem that pumps the liquid sheath flow from the sheathliquid reservoir into the viewing zone and a waste sheath liquid pumpthat pumps waste sheath liquid from the viewing zone into the wastetank. Although it appears that the disclosed sheath liquid subsystem hasnever been used in velocity critical flow cytometers, this patentreports that the disclosed sheath liquid subsystem overcomes most of thedrawbacks of conventional sheath liquid flow stabilization by:

1. damping pump pulsations by locating:

-   -   a. one fluidic capacitor between the sheath liquid pump and the        flow cell; and    -   b. another fluidic capacitor between the flow cell and the waste        pump; and

2. a pump controller whose operation is responsive to a pressure sensorthat measures the pressure difference between the inlet and outlet ofthe flow cell.

However, the disclosed sheath liquid subsystem has other limitations.For example, the pressure sensor located near the outlet of the flowcell could be a potential source of contamination.

FIG. 14 depicts a fluidic subsystem 70 in accordance with someembodiments of the present disclosure that includes a sheath liquidreservoir 702 and a liquid pump 701 that draws sheath liquid from thesheath liquid reservoir 702. The liquid pump 701 may be a diaphragmpump, a peristaltic pump, a piston pump, or any types of continuousfluid pump. An outlet of the liquid pump 701 may connect to an inlet ofa T-coupling 703 that receives sheath liquid from the liquid pump 701.The T-coupling 703 may have two (2) outlets. The first outlet mayconnect to a bypass conduit 710 for returning a fraction of the sheathliquid received by the T-coupling 703 from the liquid pump 701 back tothe sheath liquid reservoir 702. Returning a fraction of the sheathliquid received by the T-coupling 703 from the liquid pump 701 back tothe sheath liquid reservoir 702 is advantageous for two (2) reasons.

1. As depicted in FIG. 14, the bypass conduit 710 is left open to thesurrounding atmosphere which effectively dampens pulsation to therebysignificantly reducing the pulsation inherent in the operation of theliquid pump 701.

2. Returning a fraction of the sheath liquid received by the T-coupling703 from the liquid pump 701 back to the sheath liquid reservoir 702also effectively reduces the throughput of the liquid pump 701 therebyallowing the use of comparatively high flow rate, low cost pumps in theflow cytometer 40.

Denote the flow resistance of the bypass conduit 710 as “r” and the flowresistance of path from the T-coupling 703 to the flow channel 604 ofthe cuvette 603 as “R.” The output resistance to the sheath pump R_(p)is then equal to:

$\begin{matrix}{R_{p}\frac{rR}{r + R}} & (1)\end{matrix}$

Since R>>r, the behavior of the liquid pump 701 is therefore dominatedby the resistance of the bypass conduit 710 whose fluid dynamicproperties may be temperature insensitive. Thus, the configuration ofthe fluidic subsystem 70 depicted in FIG. 14 may also provide a simplemechanism for achieving a temperature insensitive sheath liquid flow tothe flow channel 604. As depicted in FIG. 14, the second outlet of theT-coupling 703 connects to the flow channel 604 that extends through thecuvette 603 first via a small reservoir capsule 704 and then via afilter cartridge 705. As depicted in FIG. 15, a piece of tubing 704′,which may be, for example, about 4 ft. long may be substituted for thesmall reservoir capsule 704. During initialization of the fluidicsubsystem 70, some air becomes trapped in the filter cartridge 705 nearits inlet which as depicted in FIG. 15 is located above an outlet of thefilter cartridge 705. The air trapped in the filter cartridge 705 mayact as an additional fluidic capacitor effectively reducing tonegligible level the pulsation in sheath liquid emitted into the flowchannel 604. Due to the large fluidic resistance at the flow channel604, the air trapped inside the filter cartridge 705 becomes compressed.When the liquid pump 701 is turned off, a trapped in the filtercartridge 705 is pushed back towards the T-coupling 703 analogous to adischarging capacitor. Without the small reservoir capsule 704, some airejected from the filter cartridge 705 reaches the bypass conduit 710 dueto its low fluidic resistance, and will be pushed out of the fluidicsubsystem 70 once the liquid pump 701 is turned on again. Withoutadditional air supply, such a scenario will repeat until most of the airbecomes purged from the fluidic subsystem 70 causing the filtercartridge 705 to lose its effectiveness as a pulsation damper. Thepurpose of the small reservoir capsule 704 or the piece of tubing 704′is therefore to provide a reservoir for isolating the filter cartridge705 from the bypass conduit 710 ensuring that air trapped inside thefilter cartridge 705 remains within the fluidic subsystem 70 despiterepeated on-off operations of the liquid pump 701.

The pulsation damping effect of the trapped air near the inlet of thefilter cartridge 705 is clearly evident in the histograms depicted inFIGS. 16A and 16B. FIG. 16A depicts measured particle flight times atthe flow channel 604 when a pocket of air is trapped near the inlet ofthe filter cartridge 705. FIG. 16B depicts measured particle flighttimes at the flow channel 604 when the trapped air is purged from thefluidic subsystem 70. The result depicted in the histograms of FIGS. 16Aand 16B is made using two (2) knife edge shaped laser beams focused nearthe center of the flow channel 604 that are spaced approximately 200micrometers apart. The horizontal axis of the FIGS. 16A and 16B is theflight time a particle takes from one laser beam to the other measuredby recording the peak arrival time of light scattered from the particleat ninety degrees (90°) from the excitation beams. In both cases, theaverage flight time for particles to cross the two laser beams is thesame. As shown in FIG. 16A, when the filter cartridge 705 retains someair, all particles take about the same amount of time to cross the twolaser beams. If the filter cartridge 705 retains no air, as shown inFIG. 16B, the distribution of flight times not only broadens, but alsobecomes bimodal. In other words, some particles take less time whileothers take longer than average amount of time to cross the two laserbeams, a phenomenon that can be easily attributed to sheath liquidvelocity pulsation at the flow channel 604.

In the embodiments of the present disclosure discussed so far, thefluidic resistance along bypass conduit 710 as well as between theT-coupling 703 and the flow channel 604 may not be adjustable. As shouldbe apparent to those ordinary skilled in the art, flow restrictors sucha fixed restrictor or adjustable valves 712, 712′ and 711, 711′ and maybe advantageously inserted in the bypass conduit 710 and between theT-coupling 703 and the flow channel 604 to permit adjusting the flowrate through the flow channel 604. Alternatively, the velocity of sheathliquid flowing through the flow channel 604 may also be adjusted using aliquid pump 701 that is driven by a variable speed brushless DC motor.

Peristaltic Pump 80

Peristaltic pumps are volumetric pumps in which a set of linearly orcircularly moving rollers progressively compress a compressible tube topropel the fluid through the tube. Peristaltic pumps are widely usedparticularly to pump clean/sterile or aggressive fluids to avoid crosscontamination with exposed pump components. Conventional peristalticpump exhibits a pulsation. Each time a roller rolls off the tube nearthe pump outlet, caused by the temporary increase of tube volume whenthe compressed tube expands back to its original shape. The pulsation isundesirable in applications that require smooth flow. Many attempts havebeen made in the past to reduce the pulsation. For example, U.S. Pat.Nos. 3,726,613 and 3,826,593 introduced a cam operated pusher whichsynchronously exerts an external pressure on the tube to compensate forthe tube expansion. In U.S. Pat. No. 4,834,630, a plurality of tubesmounted on segmented rollers are joined together at the pump inlet andoutlet by T-shaped couplers such that pulsations from individual tubeswould be reduced by averaging. U.S. Pat. No. 7,645,127 proposed a pumptube with slightly larger inner diameter near the inlet so that the tubedecompression near the pump outlet is compensated by the compression ofa larger volume tube near the inlet. The various methods eithersignificantly increased the complexity of the peristaltic pump or hadlimited success in reducing the pulsation effect.

A peristaltic pump 80 in accordance with some embodiments of the presentdisclosure is illustrated in FIG. 17. The pump may include a housing 809with arcuate curved track 808, three rollers 810, 811 and 812 attachedto a rotor 816 rotatable within the housing 809, and a compressible tube807 sandwiched between the arcuate curved track 808 of the housing 809and the rollers 810, 811 and 812, in particular at the surface 814 ofrollers 810, 811 and 812. As depicted schematically in FIGS. 18A through18D, the rollers 810, 811 and 812 of the peristaltic pump 80 are spacedat substantially equal angular distances, separations or spacings fromeach other around the perimeter of the rotor 816. The rollers 810, 811,812 may rotate about a longitudinal axis thereof, so that limitedfriction occurs between the rollers and the compressible tube. This mayalso apply for the subsequently described rollers. For simplicity, it isassumed in the following discussions that the rotor 816 rotatescounterclockwise, although it is to be understood that the discussionsapply equally well to a peristaltic pump with clockwise rotating rotor.The compressible tube 807 of the housing 809 may be divided into severalsections:

1. an open section between point 801 and point 806 where thecompressible tube 807 experiences no compression;

2. a pump inlet section between point 801 and point 802 where thecompressible tube 807 is progressively compressed until fully closedwhen a roller rolls over the section;

3. two pumping sections between point 802 and point 803, as well asbetween point 804 and point 805 wherein the compressible tube 807 isfully closed by the roller;

4. a recess section between point 803 and point 804 in which thecompressible tube 807 progressively expands from fully closed to fullyopen as a roller rolls through the expansion part of the recess sectionfrom point 803 to point 813;

5. then the compressible tube 807 is progressively compressed to fullyclosed as a roller rolls through a compression part of the recesssection from point 813 to point 804; and

6. the exit section between point 805 and point 806 where thecompressible tube 807 progressively expands from fully closed to fullyopen as a roller rolls through the section.

In other words, when a roller rolls anticlockwise over the compressibletube 807 from inlet point 801 to outlet point 806, the inner gap of thecompressible tube 807 may:

1. progressively decrease from fully open at point 801, to fully closedat point 802 and remain closed until point 803;

2. then progressively expand back to fully open at point 813;

3. then progressively decrease to fully closed at point 804, and remainclosed until the roller reaches point 805; and

4. finally progressively expand back to fully open at point 806.

The size of the gap inside the compressible tube 807 is schematicallyillustrated in FIGS. 18A through 18D as the spacing between dashedcircle and the solid compressible tube 807. As illustrated in FIGS. 18Athrough 18D, in this embodiment of the peristaltic pump 80, the angulardistances, separations or spacings between points 801 and 803, points802 and 813, point 813 and 805, as well as between point 804 and 806 maybe identical to the angle between adjacent rollers. As a result, whenthe roller 810 rolls through the pumping section from point 804 to point805, as depicted in FIGS. 18A through 18B, its interaction with thecompressible tube 807 may completely determine the fluid flow rate ofthe peristaltic pump 80. Once the roller 810 reaches the exit sectionbetween point 805 and 806, as shown in FIG. 18C, the compressible tube807 underneath the roller 810 may start to progressively expand and agap may start to grow. Meanwhile, the roller 811 may arrive at thecompression part of the recess section and start to progressivelycompress the compressible tube 807. In the peristaltic pump 80, theshape of the compression part of the recess section between point 813and point 804 along the compressible tube 807 is such that the volume ofliquid pushed out by the compression of the underneath roller 811 in thecompression part of the recess section between point 813 and point 804may substantially fill the volume created by the compressible tube 807expansion underneath roller 810 in the exit section between point 805and point 806. During this period, the compressible tube 807 ispartially open underneath both rollers 810 and 811 and completely closedunderneath roller 812. Consequently, the pumping action may be mainlydelivered by roller 812. In particular, since by design the total volumeof liquid in the section of the compressible tube 807 between point 813and point 816 remains substantially constant during this period, theflow rate of the peristaltic pump 80 in the state shown in FIG. 18C mayremain substantially the same as that in the state shown in FIGS. 18Aand 18B. Once the roller 810 passes point 806, roller 811 reaches thepumping section between point 804 and point 805. Note there is nophysical difference between the rollers 810, 811 and 812, the flow rateof the peristaltic pump 80 may therefore remain substantially constantthroughout the entire process.

The mechanism of the pulseless peristaltic pump in accordance with someembodiments of the present disclosure may be understood more clearly ifit is viewed along a circular coordinate following the movement of therollers. Referring to FIG. 19, denote as V the volume of the fluidinside a compressible tube 819 from the outlet to a nearest roller 820that closes off the compressible tube 819, i.e., the amount of fluidrepresented by the hatched area 818 shown in FIG. 19. Clearly, V dependson the angular position, θ, of the roller 820, as well as δ, the amountof tube compression exerted by all other downstream rollers.

V=V(θ, δ₁, δ₂, . . . )   (2)

Consequently, the flow rate, F, of a peristaltic pump is related to thetime derivative of V_(c) by:

$\begin{matrix}{{- F} = {\frac{dv}{dt} = {{\frac{\partial v}{\partial\theta}R} + {\sum_{i}{\frac{\partial v}{\partial\delta_{i}}\frac{d\; \delta_{i}}{dt}}}}}} & (3)\end{matrix}$

Here R is the rotational speed of the rotor and the subscripts are usedto identify multiple downstream rollers. The first term on the righthand side of Eqn. (3) represents the

contribution from the roller that closes off the tube. The partialderivative

$\frac{\partial v}{\partial\theta}$

is therefore independent of θ. The summation term representscontributions from all other downstream rollers partially compressingthe compressible tube 819. Now let ΔS be the cross sectional area changedue to the compression of the compressible tube 819 by the roller 817,and L be the length of tube where its cross sectional shape is affectedby the tube compression. Then, it is obvious to a person skilled in theart that L is proportional to the tube compression δ, and ΔSproportional to its square, δ². Consequently, ΔV, the volume of fluidlost due to the compression of the compressible tube 807 by the roller,follows Eqn. (4):

ΔV αL·ΔSαδ ³=(D−G)³   (4)

where D is the inner diameter of the compressible tube and G is theminimum gap indicated in FIGS. 19, 19A and 19B which is also representedin FIGS. 18A through 18D by the spacing between the dashed circle andthe solid track 808 of the housing 809. FIG. 19A and 19B are detailedcross-sectional views orthogonal to the peristaltic pump's tube's lengthtaken along the lines 19A and 19B in FIG. 19 illustrating the tube'spartial compression by the roller. FIG. 19A shows the cross-sectionalarea 818 a taken along the line 19A in FIG. 19. FIG. 19B shows thecross-sectional area 818 b taken along the line 19B in FIG. 19. Nowreferring to FIGS. 20A and 20B, in the circular coordinate system, FIG.20A corresponds to the state of pump shown in FIGS. 18A and 18B. Duringthis period, there is no roller downstream of the roller 810′ and thesummation term in Eqn. (3) vanishes. FIG. 20B corresponds to the stateof the pump shown in FIG. 18C. The compressible tube 807 is closed offby roller 812′ and partially compressed by the rollers 810′ and 811′.However, volumetric changes introduced by the two rollers 810′ and 811′substantially cancel each other. Consequently, the summation term isEqn.(3) vanishes as well. As a result, the flow rate of the peristalticpump 80 may remain substantially constant regardless of rollerpositions.

The shape of the compressible tube 807 satisfying the above requirementcan be readily derived from Eqn. (4). Referring to FIG. 18C, if the gapsof the arcuate curved track 808 in the compression part of the recesssection between point 813 and point 804, G_(13,4), and in the exitsection between point 805 and point 806, G_(5,6), follow the equation:

(D−G _(13,4))³+(D−G _(5,6))³ =D ³   (5)

then the total fluid volume in the two sections may remain substantiallyconstant, as shown in FIG. 21. In the peristaltic pump 80, the shape ofthe pump housing 809 may be symmetrical with respect to its center line,such that the entrance half of the pump housing 809 is the mirror imageof the exit half of the housing 809, as shown in FIG. 17. Theperistaltic pump 80 can therefore be operated both in counterclockwiseand clockwise rotation with very little pulsation, although it isunderstood that the symmetry is not required to realize a pulselessperistaltic pump in accordance with some embodiments of the presentdisclosure. For example, as long as the gaps of the arcuate curved track808 in the section between point 813 and point 803, G_(13,3), and in thesection between point 802 and point 801, G_(2,1), follow Eqn. (6)

(D−G _(13,3))³+(D−G _(2,1))³ =D ³   (6)

a peristaltic pump in accordance with some embodiments of the presentdisclosure will exhibit little pulsation when the rotor 816 rotatesclockwise.

FIG. 22 depicts an alternative embodiment of a peristaltic pump inaccordance with some embodiments of the present disclosure. Thoseelements depicted in FIG. 22 that are common to the peristaltic pump 80illustrated in FIG. 17 carry the same reference numeral distinguished bya prime (′) designation. The peristaltic pump 80′ may include an arcuatecurved track 808′ having two (2) recesses 818 and 819, and four (4)rollers 820, 821, 822 and 823. In the embodiment depicted in FIG. 22,the fluid volume loss due to the tube expansion near the outlet of thepump is compensated by the combined effect of the compression of thecompressible tube by rollers 820 and 823 near the two recesses 818 and819.

FIG. 23 depicts yet another alternative embodiment of a peristaltic pumpin accordance with the present disclosure. Those elements depicted inFIG. 23 that are common to the peristaltic pump 80 illustrated in FIG.17 and the peristaltic pump 80′ illustrated in FIG. 22 carry the samereference numeral distinguished by a double prime (″) designation. Theperistaltic pump 80″ may include six (6) rollers 820, 821, 822, 823,824, 825 and an arcuate curved track 808″ having two recesses 818″ and819″. In the peristaltic pump 80″, the fluid volume loss due to the tubeexpansion near the outlet of the pump is compensated by the action ofthe roller immediately upstream of the one recess 818″ or 819 ″ near thepump outlet.

Pulsation due to the expansion of a compressed compressible tube nearthe outlet of a peristaltic pump may also be overcome by a peristalticpump having a programmable rotor speed. FIGS. 24A through 24C illustratepertinent aspects of an alternative embodiment mechanism for minimizingperistaltic pump pulsation in accordance with the present disclosure fora 3-roller peristaltic pump. As depicted in FIG. 24B, the track 828 issubstantially circular between the pump inlet and pump exit section.Consequently, as indicated by the spacing between the dashed circle 829and the solid curve of the track 828, the compressible tube iscompletely closed by various ones of the pump's three (3) rollers 825,826, 827 between the pump inlet and pump outlet. FIG. 24A illustratesschematically in a circular coordinate system the roller positions forthe peristaltic pump depicted in FIG. 24B. Since there is only oneroller downstream of the one that closes off the tube, Eqn. (3) is muchsimplified:

$\begin{matrix}{{- F} = {R\left( {\frac{\partial V_{c}}{\partial\theta} + {\frac{\partial V_{c}}{\partial\delta}\frac{d\; \delta}{d\; \theta}}} \right)}} & (7)\end{matrix}$

Here the tube compression δ (θ) is explicitly expressed as a function ofroller position θ. The terms inside the parentheses represent the changerate of fluid volume with respect to roller position. The first term isthe contribution from the roller that closes off the tube, i.e., roller827 in FIG. 24A, and the second term the contribution from the roller inthe exit section. Note that by definition, the volume change rate isnegative and the second term inside the parentheses vanishes when thereis no roller in the exit section. The dotted curve in FIG. 24C is arepresentative plot of the negative volume change rate with respect tothe position of the roller. The bump along the curve, due to the tubeexpansion when a roller rolls off the tube near the pump outlet, is thecause for pulsation in conventional peristaltic pumps having a constantrotor speed. However, for the peristaltic pump depicted in FIGS. 24Athrough 24C, the rotor speed, R, shown as dashed curve in FIG. 24C, maybe set to vary in synchronism with the rotor position and inverselyproportional to the change rate of the fluid volume. Consequently, theflow rate of the pump, which is the product of the rotor speed and thechange rate of the fluid volume, may remain constant, as indicated bythe solid line at the top of FIG. 24C. Note the terms inside theparentheses of Eqn. (7) may be uniquely determined by the mechanicalstructure of the pump. The rotor speed profile can therefore be readilygenerated from the shape of the track 828 in accordance with Eqn. (4).

To those skilled in the art, there are many ways to realize aprogrammable rotor, for example, with stepping motor or DC servo motor.

WDM Device 90

In many multicolor fluorescence detection instrumentations, such as flowcytometers, (Practical Flow Cytometry, Howard M. Shapiro, Wiley (2003)ISBN 0471411256), the fluorescence light emitted from the object ofinterest is:

1. collected by a microscope objective;

2. reimaged through a small pinhole or a multimode optical fiber;

3. then collimated and separated into multiple colored bands; and

4. finally detected by photo detector, such as photomultiplier tube(PMT), PIN photodiode or avalanche photodiode (APD).

A PMT is essentially a special type of electron tube. This“pre-semiconductor age” device is bulky and expensive. In addition, ithas poorer quantum efficiency and less reproducible spectral responsethan silicon based semiconductor detectors, particularly in thebiologically important red to near infrared spectral region. Despite thedisadvantages, PMT has excellent noise characteristics. For example, thedark current of a typical 13 mm PMT (e.g., the R9305 from HamamatsuCorporation of Japan) is only about 1 nA. In contrast, an APD's darkcurrent would be 10 times greater even if its active area were reducedto 1/20th of that of the PMT. As a result, PMT has been the de-factolow-level light detector in many commercial fluorescence detection flowcytometers. Only in certain scientific applications where event rate islow and dark current may be discriminated against by expensivephoton-counting techniques that the PMT has been replaced by APDdetectors. (c.f., High-Throughput Flow Cytometric DNA Fragment Sizing,A. V. Orden, R. A. Keller, and W. P.Ambrose, Anal. Chem., 2000, 72 (1),p 37-41). More recently, a Geiger mode APD array was also promoted asPMT replacement. (For example, the multi pixel photon counter ofHamamatsu Photonics of Japan and the solid-state photomultiplier ofSensL Inc. of Ireland.) These detectors, however, also have high darkcurrent and are nonlinear at high event rate.

The only industry where APD has found wide acceptance is in opticalcommunication. It is known that if the APD's active area is reduced toless than 1 mm², the corresponding dark current will be reduced to thesame level as a PMT. In optical communication, the light is a laser beamout of single mode optical fiber. Such a beam can be easily collimatedthen focused down to an area much smaller than 1 mm². It should be notedthat the color separation devices used in the fluorescence lightdetection instruments, as described in U.S. Pat. No. 6,683,314 andreferences therein, are almost identical in function and architecture tothe wavelength division multiplexers (WDM) widely used in opticalcommunication, as described in U.S. Pat. Nos. 4,482,994, and 5,786,915.A fundamental reasons preventing the use of small area APD influorescence detection instrumentation is the well-known theorem ofetendue conservation: the fluorescence light coming through a pinhole ormultimode optical fiber is an extended light source with an etenduehundreds of times greater than that of a laser beam out of a single modeoptical fiber. Consequently, as illustrated in FIG. 26, it cannot becollimated over an extended distance unless the diameter of the beam issignificantly expanded. Unfortunately, the larger the beam diameter, thegreater the technical challenge to focus it down to a small spot. Sinceefficient color separation can only be accomplished economically withcollimated light beam, small area APD has not been considered viable formulticolor fluorescence light detection applications. Clearly, atechnology capable of collimating a large etendue light beam over anextended distance without significantly expanding the beam diameterwould be highly desirable. Such a technology would enable a WDM likedevice for fluorescence light detection with characteristics comparableto low noise semiconductor detectors.

FIG. 25 shows the optical ray trace for an exemplary 6 port wavelengthdivision multiplexer of the present disclosure using zig-zagconfiguration. As shown in FIG. 25, fluorescence light going through apinhole or emitted from the facet of a multimode optical fiber, such asthe optical fiber 852 depicted in FIG. 1, forms an extended object orlight source at location 901, i.e. the optical input of the WDM 90. Thesize of the object is defined by the diameter of the pinhole or the corediameter of the multimode optical fiber. Note that the practical size ofthe pinhole or the core diameter of the multimode optical fiber ismeasured in millimeters, in contrast to the diameter of single modeoptical fibers that are measured in micrometers. Consequently, theetendue of the fluorescence light source, defined as the product of beamsize and its divergence angle, is hundreds times greater than itscounterpart in optical communication. According to the theorem of theconservation of etendue (Julio Chaves, Introduction to NonimagingOptics, CRC Press, 2008 [ISBN 978-1420054293]), light from such anextended source, similar to that from a flash light, can only be keptcollimated for a very limited distance, particularly when the diameterof the collimated portion needs to be small.

As depicted in FIG. 25, a collimating optical element, in this case anachromatic doublet lens 902, may capture the light from source 901, andproject a magnified image of the object near a final focusing lens 905.The size of the image near 905 may be kept approximately the same as theeffective size of the collimating optical element 902. Consequently,beam of light propagating between the collimating optical element 902and the focusing lens 905 may be effectively collimated. As shown inFIG. 25, so long as the magnification factor is kept small, for example,less than around 10, using a simple singlet lens as the focusing lens905, the collimated beam of light can readily focused down to a spotsmaller than that of the beam of light received by the WDM 90 atlocation 901. The ability to focus the beam of light down to such asmall size permits placing a small area semiconductor detector at afocal point 906 of the focusing lens 905 for efficient photo detection.

A dichroic filter 903, oriented at a slanted angle, may be inserted intothe optical path in between the collimating optical element 902 and thefocusing lens 905. The dichroic filter 903 may pass the color band ofinterest and reflects the remaining colors in the beam of light forfurther processing within the WDM 90. An optional band pass filter 904may be inserted following the dichroic filter 903 to further improve thecolor isolation capability of the WDM 90.

Light reflected from the dichroic filter 903 may impinge upon a secondoptical element 907, such as a concave mirror. The concave mirror 907may a radius of curvature approximately equal to the distance betweenthe collimating optical element 902 and the image near focusing lens905. The concave mirror 907 therefore creates a second image of thecollimating lens 902 near a second focusing lens 908. The light beambetween the concave mirror 907 and the second image at the lens 908 mayhave substantially the same diameter as the beam of light between thecollimating lens 902 and the first image near the focusing lens 905. Therelay imaging concave mirror 907 therefore effectively doubles thecollimated beam path without expanding the beam's diameter. Again, theextended yet collimated beam can be easily focused down to a spotsmaller than that of the light source at 901. The diameter of the spotmay be smaller than 1 mm, for example, around 600 μm, A second dichroicfilter 909 may then be inserted in between the relay imaging concavemirror 907 and the second image near focusing lens 908. The seconddichroic filter 909 may pass another band of color in the beam of lightreceived by the WDM 90 at location 901 and reflect the remainder of theimpinging beam of light for further processing. The first and seconddichroic filters 903 and 909 may be inserted approximately midwaybetween the collimating optical element 902 and the focusing lens 905and between the relay imaging concave mirror 907 and the second imagenear focusing lens 908, respectively.

As shown in FIG. 25, additional relay collimating optical elements 910,911, 912, 913 and dichroic filters 914, 915, 916, 917 can be cascaded inthe same way to produce multiple images near focusing lenses 918, 919,920 and 921, each of these images corresponding to a specific color bandof light received by the WDM 90 at location 901. As shown in FIG. 25,due to the present disclosure's 1:1 imaging relay architecture, thespots of light produced by focusing lenses 905, 908, 918, 919, 920 and921 are all smaller than the source of the beam of light, and thereforecan be easily captured by small area APD's.

Although FIG. 25 illustrates a 6-port wavelength division multiplexerfor a beam of light from the extended light source, it is readilyapparent to those skilled in the art that WDM's having different numbersof ports can be easily built in accordance with some embodiments of thepresent disclosure. It is also apparent to those skilled in the art thatalthough the WDM 90 may use achromatic doublets as the first collimatingoptical element, singlet lens can also be used since the images createdbefore the focusing lenses 905, 908, 918, 919, 920 and 921 are allnearly monochromatic. Instead of using concave mirrors for relaying thebeam of light reflected from the dichroic filters, one may also userefractive optics, such as a convex lens, as a relay element to extendthe path of the collimated beam of light. One of the advantages of thezig-zag architecture used in the WDM 90, however, is the possibility forusing array detectors, which would lead to a more compact WDM suitablefor portable instrumentation.

FIG. 25A illustrates a top view of a light detection assembly 937 withthe WDM 90 illustrated in FIG. 25 in accordance with some embodiments ofthe present disclosure. The light detection assembly 937 may include theWDM 90 and a photodetector system 938. A beam of light which emits fromthe facet of the optical fiber 852 may be processed by the WDM 90 anddetected by the photodetector system 938. The concave mirrors 907, 910,911, 912, and 913 and the dichroic filters 903, 909, 914, 915, 916, and917 may be formed on two sides of a reference block 935. The referenceblock 935 may be made of glass or any material which allows light topass through it. Accordingly, a zig-zag optical pattern, as illustratedin FIG. 25 may be formed among the collimating optical element 902, thedichroic filters 903, 909, 914, 915, 916, and 917, the reference block935, the concave mirrors 907, 910, 911, 912, and 913, and the focusinglens 905, 908, 918, 919, 920, and 921. After being processed by the WDM90, the beam of light emitting from the facet of the optical fiber 852may split into multiple colored bands with different wavelengths to bedetected by photodetectors 940, 941, 942, 943, 944, and 945,respectively. The photodetector can be, but is not limited to, asemiconductor detector, an avalanche photodetector (APD), and a carbonnanotube detector.

In some embodiments, the concave mirrors 907, 910, 911, 912, and 913 maybe structurally formed on a relaying assembly 939. It should beunderstood by those having skill in the art that the concave mirror canbe replaced with a convex lens, which is also able to converge and relaythe beam of light.

In some embodiments, the dichroic filter can be replaced with a mirrorto prevent the beam of light from entering a photodetector when a userwants to decrease the number of light signal channels to be detected. Itshould be understood by those having skill in the art that the dichroicfilter can also be replaced by a dichroic mirror, a beam splitter, orany optical element which is able to split or filter a beam of light.

FIG. 25B illustrates a front view of the light detection assembly 937with the WDM 90 illustrated in FIGS. 25 and 25A in accordance with someembodiments of the present disclosure. The light detection assembly 937may have a top cover 940 which is openable. Therefore, the user can openthe top cover 940 to change the dichroic mirrors 903, 909, 914, 915, 916and 917 and modify the light detection system 938 or the WDM 90 inside.

FIG. 26 illustrates an optical ray trace for a prior art collimatingdevice. The technique depicted in FIG. 26 is extensively used inconventional multicolor fluorescence instruments, for example, in U.S.Pat. No. 6,683,314. As shown in FIG. 26, the beam of light divergesrapidly beyond the image 924 created by the collimating optical element923. Consequently, the only option for constructing a multi-color deviceis to insert dichroic filters in between the collimating element 923 andits image 924.

Due to the constraint of etendue conservation, the diameter of thecollimated beam must be significantly expanded to accept multipledichroic filters in the section. The expanded beam creates seriouschallenge to refocusing the collimated beam down to small spots suitablefor small area semiconductor detectors. To overcome these difficulties,some instrument manufacturers have chosen to use PMT exclusively forfluorescence detection such as in the main stream flow cytometersmanufactured by Becton-Dickinson, Beckman Coulter and Partec's and theMegaBACE series of DNA sequencers by GE Amersham. Other instruments,such as the Luminex multiplexed bead analyzers, have selected certaincolor bands with known bright fluorescence, and uses large area APD fordetecting light in the selected color bands.

FIG. 27 illustrates a perspective view of an alternative embodiment fora 6-port WDM 90 using a combination of zig-zag and branchedconfigurations. The design is a modification of zig-zag configurationdepicted in FIG. 25. In the alternative embodiment depicted in FIG. 27,band pass filter 904 of FIG. 25 may be replaced by a dichroic filter904′. The filter 904′ is positioned to let one color pass through andreflects other colors at ninety degrees (90°). The optical path lengthof the beam of light passing through the dichroic filter 904′ and thatbeing reflected from the 904′ are substantially the same, such that onearm is focused by lenses 905 and the other by lens 905′ to small spotscompatible with small area semiconductor detectors placed at focallocation 906 and 906′. As shown in FIG. 25, the remaining color of thelight reflected by dichroic filter 903 is relay imaged by a concavemirror 907 and the configuration including optical elements 903, 904′,905 and 905′ is cascaded two (2) more times to form a 6 port-WDM.

FIG. 28 illustrates a perspective view of an alternative embodiment fora 8-port WDM 90. By replacing the concave mirrors 907 and 910 in FIG. 27with concave shaped dichroic filters 907′ and 910′, the WDM depicted inFIG. 28 may provide 2 more color bands in comparison with the WDMsdepicted in FIGS. 25 and 27.

Numerous fluorescence probes for use in flow cytometry have beendeveloped over the years. More recently, multiple fluorescence proteinshave also become an important tool in biomedical studies. To accommodatedifferent types of fluorescence probe, various techniques have beendeveloped to enable user selection of dichroic filters suitable fortheir particular needs. A significant challenge for replaceable dichroicfilters is avoiding direct contact of the coated filter surface with anyhard flow cytometer reference frame. Repeated direct contact between thecoated filter surface and any hard reference frame may damage areplaceable dichroic filter. Presently, most conventional solutionsaddressing this problem use precision-machined mechanical spacers forholding replaceable dichroic filters in place. One example of such asolution appears in U.S. Pat. No. 6,683,314. However, such a solutionbecomes unreliable if the detector's active area is smaller than 1.0mm².

FIGS. 29A and 29B depict fabricating a replaceable dichroic filterassembly 934 illustrated in FIG. 29C suitable for small area detectors.Assembly of the replaceable dichroic filter assembly 934 begins in FIG.29A which depicts constructing a reference template for its fabrication.The reference template may be a staircase made of two (2) opticallyparallel glass plates 925 and 926. Bonding the two (2) glass plates 925and 926 together in optical contact can ensure that a surface 929 of theglass plate 925 becomes optically parallel to a surface 930 of the glassplate 926. A front surface 932 of a replaceable dichroic filter 927 maythen be pressed against the surface 929 of the template. A filter holder928, which loosely fits the dichroic filter 927, may include a referencesurface 931 and a filter slot 933. During assembly of the replaceabledichroic filter, the filter slot 933 may be partially filled with epoxyadhesive and the reference surface 931 of the filter holder 928 may bepressed against the surface 930 of the template while filter holder 928slides toward the dichroic filter 927. While the epoxy adhesive sets,part of the dichroic filter 927 remains seated within the filter slot933 while pressure is applied against the dichroic filter 927 and filterholder 928. It should be apparent to those skilled in the art that theepoxy adhesive may be either UV or thermally curable, or made byblending together components of an AB mixture. FIG. 29C depicts adichroic filter fabricated as depicted in FIGS. 29A and 29B anddescribed above. The assembly process depicted in FIGS. 29A and 29B anddescribed above can ensure that the front surface 932 of the replaceabledichroic filter assembly 934 can be optically parallel to the referencesurface 931, and indented with respect to the latter at a spacingaccurately determined by the thickness of the glass plate 925. Thedichroic filter 927 depicted in FIGS. 29A, 29B, and 29C may be thedichroic filters 903, 909, 914, 915, 916, or 917 used with the WDMillustrated in FIG. 25 or the dichroic filter 903 or the filter 904′used with the WDM illustrated in FIG. 27.

FIGS. 30A and 30B depict an embodiment of the present disclosure wherethe fore-mentioned replaceable dichroic filter assembly 934 is used inthe WDM 90 for optically processing a beam of light from an extendedlight source. A notable feature of the WDM 90 is a glass reference block935 having an optically flat surface. As will be apparent to thoseskilled in the art, the glass reference block 935 may be made of othermaterials. As shown in FIG. 30B, when installing a dichroic filter 927the reference surface 931 of the replaceable dichroic filter assembly934 may slide against the flat surface of the glass reference block 935and be kept in contact therewith by a spring loaded screw 936.Consequently, the coated front surface 932 of the replaceable dichroicfilter assembly 934 can remain optically parallel to the optical flatand accurately located. In the meantime, the indentation of frontsurface 932 with respect to the reference surface 931 may protect itfrom in physical contact with any object during filter replacement. Itis apparent to those skilled in the art that many modifications andvariations of the described embodiments of the replaceable dichroicfilter assembly 934 are possible. For example, an alternative embodimentof the present disclosure may be a pedestal assembled using a first anda second round optical flat. When assembling the replaceable dichroicfilter assembly 934, the reference surface of a filter holder may restagainst a surface of the first optical flat and the coated surface ofthe dichroic filter may rest against the flat surface of the secondoptical flat. Epoxy bonding then may hold the coated surface of thedichroic filter optically parallel to the reference surface of a filterholder, yet indented at a distance accurately determined by thethickness of the second optical flat.

Optical System with Single Light Source 41

FIG. 31 is a diagram schematically illustrating an optical system with asingle light source 41 in accordance with some embodiments of thepresent disclosure. The optical system with a single light source 41 mayinclude a LD based optical subsystem 50, a composite microscopeobjective 60, a WDM 90, and a light detection system 938. The beam oflight may propagate substantially along the z axis and enter thecomposite microscope objective 60 from the LD based optical subsystem 50to illuminate particles present within the viewing zone inside thecomposite microscope objective 60. The light scattered from andfluoresced by particles may then be reflected by the concave mirror 601,corrected by the corrector plate 602, and collected by the optical fiber852 substantially along the x axis. The optical fiber 852 may be fixedby a fiber holder 940.

Common wavelengths of light sources may include, but not limited to, 375nm, 405 nm, 440 nm, 488 nm, 502 nm, 534 nm, 561 nm, 591 nm, 637 nm, and637nm. The light detection system 938 may be coupled with circuits forprocessing light signals. The more ports the WDM 90 has, the more lightsignal channels the user can use.

Optical System with Multiple Light Sources 42

FIG. 32 is a diagram schematically illustrating an optical system withmultiple light sources 42 in accordance with some embodiments of thepresent disclosure. The optical system with multiple light sources 42may include multiple LD based optical subsystems 50, multiple WDMs 90,multiple light detection systems 938, and a composite microscopeobjective 60 with a viewing zone. The number of WDMs 90 may correspondto the number of LD based optical subsystems 50. In FIG. 32, the opticalsystem with multiple light sources 42 includes three laser diodes 501for emitting multiple beams of light with different wavelengths, threecollimating lenses 502 in front of the three LDs for collimating thebeams of light respectively, three dichroic filters 506, 507, and 508for passing beams of light with certain wavelength range or reflectingbeams of light with certain wavelength range, a plano-convex lens 504for shaping the beams of light on the major axis, a cylindrical lens 505for focusing the beams of light onto three spatially separated locationsin the flow channel 604, a composite microscope objective 60 fordirecting the light scattered from and fluoresced by the illuminatedparticles at three spatially separated locations to be collected bythree optical fibers 852, respectively, three optical fibers 852 forcollecting scatter and fluorescence emissions and transmitting theemissions to three WDMs 90, respectively, and three WDMs 90 and lightdetection systems 938 for processing and detecting the scatter andfluorescence light, respectively. The direction of beams of lightentering the composite microscope objective 60 may be perpendicular tothe direction of scatter and fluoresce emissions to be collected by theoptical fiber 852. It should be noted that the plano-convex lens 504 andthe cylindrical lens 505 can be replaced with any conventional beamshaper and any focusing lens. It should also be noted that the variousaspects of the present disclosure are not limited to specific numbers oflaser diodes, collimating lenses, dichroic filters, plano-convex lenses,composite microscope objectives, optical fibers, WDMs, and lightdetection systems and specific wavelength and direction of each beam oflight.

FIG. 33 illustrates an enlarged view of beams of light 509 and 510 shownin FIG. 32. The beams of light 509 and 510 are emitted from differentlaser diodes 501 with different wavelengths and then are focused ontospatially divided locations in the flow channel 604 inside the compositemicroscope objective 60.

Optical System with Chromatic Compensation Elements 51

FIG. 34 is a diagram schematically illustrating an optical system withchromatic compensation elements 51 in accordance with one aspect of thepresent disclosure in accordance with some embodiments of the presentdisclosure. The optical system with chromatic compensation elements 51may include the optical system with multiple light sources 42, as shownin FIG. 32, and multiple chromatic compensation elements 514, 515, and516. Each of the chromatic compensation elements 514, 515, and 516 maybe positioned on the beam paths of beams of light emitting from lightsources 511, 512, and 513, respectively, and compensate chromaticaberration in the viewing zone. As such, the beams of light emittingfrom the light sources 511, 512, and 513 with different wavelengths maybe focused onto three spatially divided locations on a common planewhich is in the viewing zone and substantially parallel to the directionof a sample flow. The optical properties of chromatic compensationelements 514, 515, and 516 may be different from each other. Forexample, their thicknesses and shapes may be different to accommodatevarious beams of light with different wavelengths.

In some embodiments, the optical system shown in FIG. 34 may only needone or two chromatic compensation elements to compensate chromaticaberration in the viewing zone. It should be noted that the variousaspects of the present disclosure are not limited to specific numbers oroptical properties of chromatic compensation elements.

Power Monitoring System 43

FIG. 35 is a diagram schematically illustrating a power monitoringsystem 43 in accordance with some embodiments of the present disclosure.The power monitoring system 43 may include a first light source 513 foremitting a first beam of light, a second light source 512 for emitting asecond beam of light, a first dichroic filter 519 for reflecting thefirst beam of light and passing the second beam of light, a seconddichroic filter 518 for reflecting the second beam of light, a firstdetector 401 for measuring residual power of the first and second beamsof light downstream of the first dichroic filter 519 on a time-divisionmultiplexing basis, and a control unit 522 coupled with the firstdetector 401, the first light source 513, and the second light source512. The first detector 401 may be positioned near or coupled to thefirst dichroic filter 519. The first and second light sources 513 and512 may emit beams of light with different wavelengths.

In order to reduce interference between the residual power of the firstand second beams of light, the first detector 401 may measure theresidual power of the first beam of light when the second light source512 is off or measure the residual power of the second beam of lightwhen the first light source 513 is off. The residual power of the firstand second beams of light may include power of the first beam of lightpassing through the first dichroic filter 519 and power of the secondbeam of light reflected by the first dichroic filter 519.

In some embodiments, the control unit 522 may include a feedback circuitto increase the power of the light source when residual power of thelight source drops below a certain level or to lower the power of thelight source when the residual power of the light source increases abovea certain level.

In some embodiments, a second detector 400 may be applied with the powermonitoring system 43 and positioned near or coupled to the seconddichroic filter 518 to measure the residual power of the second beam oflight downstream of the second dichroic filter 518. The residual powerof the second beam of light downstream of the second dichroic filter 518may include power of the second beam of light passing through the seconddichroic filter 518. The second detector 400 may also be coupled to thecontrol circuit 522. When the second detector 400 is applied to thepower monitoring system 43, the first detector 401 may only need tomonitor the residual power of the first beam of light.

In some embodiments, a third light source 511 for emitting a third beamof light and a third dichroic filter 517 for reflecting the third lightmay be also applied with the power monitoring system 43. The third lightsource 511 may be also coupled to the control circuit 522. As such, thefirst detector 401 may measure residual power of the first, second, andthird beams of light downstream of the first dichroic filter 519 on atime-division multiplexing basis.

In some embodiments, the second detector 400 may measure residual powerof the second and third beams of light downstream of the second dichroicfilter 518 on a time-division multiplexing basis. The residual power ofthe second and third beams of light downstream of the second dichroicfilter 518 may include power of the second beam of light passing throughthe second dichroic filter 518 and power of the third beam of lightreflected by the second dichroic filter 518.

In some embodiments, a third detector (not shown in FIG. 35) may also beapplied with the power monitoring system 43 and positioned near orcoupled to the third dichroic filter 517 to measure residual power ofthe third beam of light downstream of the third dichroic filter 517. Thethird detector may be also coupled to the control circuit 522. Theresidual power of the third beam of light at the downstream of the thirddichroic filter 517 may include power of the third beam of light passingthrough the third dichroic filter 517.

The second beam of light can either be detected by the first detector401 or the second detector 400. The third beam of light can be detectedby the first detector 401, the second detector 400, or the thirddetector which is positioned near or coupled to the third dichroicfilter 517. The control circuit 522 may control the operation of thedetectors and light sources.

It should be understood by those having skill in the art that thedichroic filter can also be replaced by a dichroic mirror or a beamsplitter. It should also be noted that the various aspects of thepresent disclosure are not limited to specific numbers of light sources,dichroic filters, and detectors.

Optical System 44

FIG. 36 is a diagram schematically illustrating an optical system 44 inaccordance with some embodiments of the present disclosure. The opticalsystem 44 may include a composite microscope objective 60, as shown inFIG. 8, a light source 403, and a beam splitter 402. The light source403 may emit beams of light to illuminate objects in a viewing zone,which is located in a flow channel 604 inside a cuvette 603. Thecomposite microscope objective 60 may image light scattered from andfluoresced by the objects in the viewing zone at an image plane 404external to the composite microscope objective 60. The light source 403and the image plane 404 may be located on two sides of the beam splitter402.

The composite microscope objective 60 may include a concave mirror 601and an aberration corrector plate 602 coupled to the two sides of thecuvette 603. The aberration corrector plate 602 may be an aspheric lensthat has a first zone with negative optical power and a second zone withpositive optical power radially inside the first zone. A neutral zonemay be the thinnest portion of the aberration corrector plate 602 andlocated between the first zone and the second zone. The aspheric lensmay be a plano-aspherical lens. The concave mirror may be aplano-concave back surface mirror or a front surface mirror. The concavemirror 604 and the aberration corrector plate 602 may be made of anoptically transparent material.

In FIG. 36, the beam of light emitting from the light source 403 may bereflected by the beam splitter 402 and enter into the compositemicroscope objective 60 to illuminate objects in the viewing zone. Thelight scattered from and fluoresced by objects may be reflected by theconcave mirror 604, transmit through the aberration corrector plate 602and the beam splitter 402, and form an image at the image plane 404external to the composite microscope objective 60. The light source 403may include multiple laser diodes 403 a, 403 b, and 403 c emittingmultiple beams of light with different wavelengths to illuminate objectsat multiple locations in the flow channel 604. Accordingly, multipleimages 404 a, 404 b, 404 c may be formed at the image plane 404.

In some embodiments, the locations of the light source 403 and the imageplane 404 may be swapped. Accordingly, the beam of light emitting fromthe light source 403 may transmit through the beam splitter 402 andenter into the composite microscope objective 60 to illuminate objectsin the viewing zone. The light scattered from and fluoresced by objectsmay be reflected by the concave mirror 604, transmit through theaberration corrector plate 602, be reflected by the beam splitter 402,and form an image at the image plane 404 external to the compositemicroscope objective 60.

In some embodiments, the viewing zone may be located in a jet stream ora surface of a substrate containing objects (not shown in FIG. 36). Theobjects may be delivered into the viewing zone by a fluidic system, suchas a fluidic system shown in FIG. 14 or 15.

In some embodiments, the scattered and fluoresced light imaged at theimage plane 404 may be received by a fiber (not shown in FIG. 36) whichtransmits the light to a photodetector. The scattered and fluorescedlight may be processed by a wavelength division multiplexer (WDM) (notshown in FIG. 36) before being detected by the photodetector. The WDMmay be configured as a WDM illustrated in FIGS. 25, 25A, and 25B. Thephotodetector can be, but not limited to, a semiconductor photodetector,a multi-pixel photon counter, and a carbon nanotube detector.

In some embodiments, the light source 403 may emit coherent light orincoherent light. The light source 403 can be single or multiple laserdiodes, light emitting diodes, illumination devices emitting beam oflight, or any combination of them.

In some embodiments, a chromatic compensating lens (not shown in thefigure) may be inserted between the aberration corrector place 602 andthe image plane 404 to serve to reduce the residual chromaticaberration.

Axial Light Loss Detection System 45

FIG. 37 is a diagram schematically illustrating an axial light lossdetection system 45 in accordance with some embodiments of the presentdisclosure. The axial light loss detection system 45 may include aconcave mirror 406 for reflecting light that propagates from a viewingzone and a detector 408 for measuring axial light loss produced by theobject in the viewing zone by detecting light reflected by the concavemirror 406. The light reflected by the concave mirror 406 may includeforward scattered light (F SC) and remaining light of beam of lightentering into the viewing zone from a light source 412 to irradiate theobject therein, which is so called axial light loss (ALL). The axiallight loss of the beam of light along its propagation direction mayresult from the object passing through the beam of light. The beam oflight may be blocked or absorbed by the object.

The axial light loss detection system 45 may utilize the concave mirror406 to direct both FSC and remaining light into the detector 408 inorder to determine the size of object. The FSC and remaining light mayhave the same wavelength, and therefore the signals of FSC and remaininglight detected by the detector 408 may be proportional to square of thesum of their electric fields as follows:

(E_(FSC)+E_(ALL))²   (8)

E_(FSC) represents the electric field of FSC. E_(ALL) represents theelectric field of remaining light.

On the contrary, a conventional ALL detection system disclosed in priorart usually requires a pinhole positioned along a laser beam path toblock FSC in order to detect remaining light of the laser beam.Accordingly, the signals of remaining light detected by an ALL detectoris proportional to square of its electric field as follows:

(E_(ALL))²   (9)

Further, a conventional FSC detection system disclosed in prior artusually requires a mask positioned along a laser beam path to blockremaining light of the laser beam in order to detect FSC. Accordingly,the light signals of FSC detected by a FSC detector is proportional tosquare of its electric field as follows:

(E_(FSC))²   (10)

Apparently, neither of the conventional ALL detection system norconventional FSC detection system could operate without using a pinholeor a mask.

In some embodiments, the concave mirror 406 may be an ellipsoidal mirroror a combination of a flat mirror and a lens. The detector 408 may be anaxial light loss detector to determine the size of the object.

In some embodiments, the detector 408 may be in a heterodyne modedetecting the coherent interference of FSC and the remaining light. Thewavelengths of FSC and remaining light may be the same.

In some embodiments, a light source 412 emitting beam of light may beused to illuminate the object in the viewing zone. The optical axis ofthe beam of light is substantially perpendicular to the flow directionof the object.

In some embodiments, multiple light sources 412 emitting beams of lightwith different wavelengths may be used to illuminate the objects in theviewing zone. When multiple light sources 412 are applied to the axiallight loss detection system 45, a filter 407 may be positioned upstreamof the detector 408 to separate the light irradiated by the first lightsource and reflected by the concave mirror 406 and the light irradiatedby the second light source and reflected by the concave mirror 406. Assuch, the detector 408 may measure them separately, for example, on atime-division multiplexing basis.

In some embodiments, the viewing zone may be located within a microscopeobjective 410. The viewing zone may be located in a flow channel 409, ajet stream, or a substrate. In some embodiments, a cylindrical lens 411may be coupled to the microscope objective 410 to focus beams of lightemitting from the light source 412 to the viewing zone. The optical axisof the cylindrical lens 411 is substantially perpendicular to theoptical axis of the light reflected by the concave mirror 406.

FIG. 38 is a diagram schematically illustrating an axial light lossdetection system 45 coupled with a second light detection system 413 inaccordance with some embodiments of the present disclosure. The axiallight loss detection system 45 may utilize a composite microscopeobjective 60, as illustrated in FIG. 8 The composite microscopeobjective 60 may include a second concave mirror 415 and an aberrationcorrector plate 414 located on two sides of the viewing zone of thecomposite microscope objective 60. The optical axes of the secondconcave mirror 415 and the aberration corrector plate 414 aresubstantially parallel to the optical axis of the light reflected by theconcave mirror 406. The FSC and remaining light propagating from theviewing zone may be reflected by the concave mirror 406 and detected bythe detector 408 while the side-scattered fluoresced light may bereflected by the second concave mirror 415, propagate out of thecomposite microscope objective 60 via the aberration corrector plate414, and be detected by the second light detection system 413.

In some embodiments, one or more control circuits may be coupled withone or more of the detector 408, the second light detection system 413,and the light source 412 to process detected light signals. As known byone skilled in the art, the control circuit may include an amplifier toamplify detected light signals, a noise filter to reduce noiseinterference, and a processor to process detected light signals andgenerate corresponding information regarding the properties of theobject.

Alternative Combined Microscope Objective

FIGS. 11, 12 and 13 illustrate the build-up of a composite microscopeobjective 60 adapted for imaging light scattered from and fluoresced byan object present within a viewing zone. The illustrated compositemicroscope objective comprises a viewing zone, a concave mirrorarrangement 601, 610, 617, an exit area and an illumination beam formingarrangement 505, as illustrated in FIG. 8A. It should be noted that inFIGS. 11, 12 and 13 the beam forming arrangement is not illustrated. Theviewing zone in FIG. 11 may be located in e.g. a channel 604 of acuvette 603. In FIG. 12, the viewing zone may be located e.g. along thedroplets of the jet stream 519 leaving the nozzle 518. In FIG. 13, theviewing zone maybe located e.g. in the plane of the substrate. The exitarea of the microscope objective is an area through which scatteredlight and fluoresced light impinging from an object present in theviewing zone passes, which scattered and fluorescent light is reflectedby the concave mirror of the microscope objective. The aberrationcorrector plate 602 in FIG. 11, 612 in FIGS. 12 and 618 in FIG. 13 maybe located in the exit area. It should be noted that the corrector platemay be used, in particular in combination with a spherical mirror 601,610, 617. However the corrector plate may be omitted when using aconcave mirror having already implemented a correcting shape. Thus, theexit area does not have to include a corrector, if a concave mirror hasan appropriate shape and the gain of the corrector plate is notrequired. As can be seen in FIGS. 11, 12 and 13, the viewing zone isarranged between the concave mirror arrangement and the exit area. Theconcave mirror 601, 610, 617 is arranged to reflect scattered andfluoresced light impinging from an object present in the viewing zone tothe exit area. The illumination beam forming arrangement 505 isillustrated e.g. in FIG. 8A. FIG. 8A illustrates an arrangement of FIG.11 having attached illumination beam arrangement 505 to the cuvette 603.The illumination beam arrangement 505 is arranged so that anillumination beam entering the illumination beam forming arrangement ispre-definitely formed at the viewing zone. A path of an illuminationbeam from an illumination system can be seen e.g. in FIG. 1 or 3A. FIGS.11, 12 and 13 as well as FIG. 8A illustrate that the concave mirrorarrangement, the viewing zone and the exit area are arranged along afirst axis, also referred to as x-axis. FIG. 9A illustrated that anoptical image of the viewing zone, e.g. within channel 604 in FIG. 9A isformed outside the composite microscope objective in the image plane 605with image locations 606, 607 and 608. The illumination beam formingarrangement 505 is arranged so that an illumination beam impinges theviewing zone along a second axis, also referred to as z-axis, which issubstantially perpendicular to said x-axis. The above described cuvette603 may be manufactured of an optical transparent material, wherein theviewing zone is formed in the cuvette, in particular in the channel 604of the cuvette 603. The channel extends along a third axis, alsoreferred to as y-axis being substantially perpendicular to the x-axisand the z-axis, so that a liquid flow in the channel flows along they-axis, as illustrated e.g. in FIG. 5D, wherein the viewing zone islocated within the channel. FIGS. 8 and 8A illustrated that the cuvette603 may be of rectangular cross section in a plane of the firstaxis/x-axis and second axis/z-axis. It should be noted that the crosssection of the cuvette 603 may also be of a form, that a sheath flowcovering the sample flow forces the sample flow into a rectangular crosssection. The cross section of the channel 604 may be constant along they-axis, but may also vary along the y-axis. In particular the channelmay have a focused cross section in the area of the viewing zone. Theviewing area may include a plurality of predefined viewing pointsdistributed along the y-axis for different illumination wavelengths, ascan be seen in FIG. 33, or along the z-axis, which may be a varyingfocal point varying along the z-axis, when adjusting the objective 60with respect to the illumination system 50, as will described later.Although element 505 may be allocated to the illumination system 50,element 505 may also be part of the objective 60, in particular it maybe attached to the cuvette 603. The illumination beam formingarrangement 505 is adapted to compress an illumination beam, so that theillumination beam in the viewing zone has a compressed dimension alongthe y-axis. The illumination beam forming arrangement 505 may be acylindrical lens, in particular having a cylindrical axis parallel tothe x-axis, as can be seen in FIGS. 5D and 5E. It should be noted thatthe illumination beam forming arrangement can be assembled by aplurality of optical elements, so that the arrangement 505 may not havea defined axis. The aberration corrector arrangement 602, 612, 618 maybe arranged in the exit area, as can be seen in FIGS. 11, 12 and 13. Theaberration corrector arrangement may be an aspheric lens made ofoptically transparent material. As can be seen in FIG. 9A, saidaberration corrector arrangement may have a first zone with negativeoptical power, a second zone radially inside the first zone withpositive optical power, and a neutral zone between the first zone andthe second zone. The neutral zone in FIG. 9A is thinner than each of thefirst zone and the second zone, so that light reflected from the concavemirror arrangement passing through said aberration corrector arrangementforms a focal area. Although FIG. 9A illustrates a corrector plate withconvex and concave portions, it should be noted that the positive andnegative optical power may be achieved by using different opticalmaterials at different locations of the corrector plate. The concavemirror arrangement 601, the viewing zone and the aberration correctorarrangement 602 form a reversed Schmidt camera. The concave mirrorarrangement may be formed by plane-convex lens, as can be seen in FIG.11. The concave mirror may be a plano-concave back surface mirror. Theplano-concave back surface mirror may be made from an opticallytransparent material. As can be seen in FIG. 8A a plano-surface of saidplano-concave back surface mirror is optically coupled to a flat surfaceof said cuvette. Plano-concave back surface mirror means that althoughthe optical lens body is a plano-convex lens body, the surface whenseeing into the mirror, which is the inside of the optic body, isconcave. The plano-surface of said plano-concave back surface mirror mayalso be optically coupled to said flat, transparent substrate, as can beseen in FIG. 13. Said concave mirror may also be a front surface mirror,as can be seen from FIG. 12. It should be noted that the mirror of FIG.12 can also be used in combination with a cuvette, and the mirror ofFIG. 11 can also be used for a jet stream. The concave mirrorarrangement, the aberration corrector arrangement and the illuminationbeam forming arrangement may be attached to the cuvette by at least oneof index matching gel, index matching fluid, optical adhesive materialand optical contact bonding. It should be noted that also a combinationmay be used for attaching.

As can be seen in FIGS. 1, 3A, 31, 32 or 34-38, the composite microscopeobjective 60 may be combined with or comprise an illumination system.Although not mandatory, the illumination may be an illumination systemas described above, in particular with a laser source 501, ancollimating optical arrangement 502 to form a collimated laser beam anda beam shaping arrangement 504, 505 being adapted to shape a collimatedbeam, wherein the beam shaping arrangement includes the illuminationbeam forming arrangement 505. The laser source may be a laser diode andthe collimating arrangement may be arranged with respect to the laserdiode so as to form a collimated beam. As an alternative, the lasersource may be a conventional laser with an optic arrangement to form acollimated beam of a desired cross section. The laser diode and thecollimation optical arrangement, or alternatively the conventional laserwith the optics are adapted to form a beam having an elliptical crosssection having a major axis and a minor axis, wherein the minor axis isoriented substantially along the y-axis and the major axis is orientedsubstantially along the x-axis, as can be seen in FIG. 5E. The beamshaping arrangement may include a major axis optical beam compressingarrangement 504 being adapted to compress at least the major axis of thecollimated elliptical beam. The illumination beam forming arrangement505 is adapted to compress at least the minor axis of the collimatedelliptical beam. This can be seen for example in FIGS. 1, 3A or 6.

The viewing zone may be movable along the z-axis with respect to theillumination system so as to vary a focus of the compressed ellipticalbeam within the viewing zone along the z-axis. This allows a scanningalong the z-axis. In particular this allows to sense or scan propertiesof a cell in the viewing zone at different locations. It should beunderstood, that either the objective 60 may be controllably moved orthe illumination system 50 or both. It should also be understood thatthe variation of the focus may also be achieved by moving singlecomponents of the illumination system, e.g. one of the mirrors 523 b,523 a or the element 504, as illustrated in FIG. 7. Also singlecomponents of the objective may be moved to vary the focus along thez-axis. The actuation can be conducted by e.g. piezo actuators oracoustic actuators. In particular the varying focus can be achieved by amodulation or an sinusoidal oscillation of the respective component.Thus the cuvette is movable with respect to the laser source so as tospatially vary a focus of the laser source in the channel. For thispurpose, a control unit can be provided being adapted to control themovement of components of the composite microscope objective along thez-axis so as to spatially vary a focus of the laser source in thechannel. It should be noted that likewise also a variation of the focuscan be achieved along the y-axis or even the x-axis.

Combined Wavelength Division Multiplexer (WDM) with Semiconductor PhotoDetector

FIGS. 25, 27, and 28 illustrate a wavelength division multiplexer (WDM)for separating light emitted from a light source into multiple coloredbands. The wavelength division multiplexer may comprise an imagingoptical arrangement 902, a dichroic filter arrangement 903, 904, asemiconductor photo detector 906 and a focusing optical arrangement 905.The imaging optical arrangement 902 forms a beam of light from the lightemitted from a light source 901 and produces an image of substantiallythe same size as the effective size of said imaging optical arrangement.The light source may be an outlet of an optical fiber, which fiber maytransfer detected light from the microscope objective 60 to the WDM. Thedichroic filter arrangement 903, 904 may be located between said imagingoptical arrangement 902 and said image, and separates the beam of lightinto a first branch and a second branch of distinctive colors. As can beseen in FIG. 25, the first branch travels toward element 905, whereasthe second branch travels toward element 907. The semiconductor photodetector 906 is located in the first branch behind the focusing opticalarrangement 905, which is located between the dichroic filterarrangement 903, 904 and the semiconductor photo detector 906 so as tofocus the beam of light onto the semiconductor photo detector. Lightmeans an electromagnetic wave, coherent or non-coherent, particularlyhaving a wavelength which transits the used optical elements. Inparticular, the term light is not limited to the visible part of light,e.g., light between 380 nm and 780 nm. It should be noted that alsoinfrared light and ultraviolet light may be used, if the used opticalcomponents are capable of being operated with such wavelengths. Thefocusing optical element arrangement 905 is located in or in proximityto an image plane of said image. It should be noted that the imagingarrangement 902 as well as the focusing arrangement 905 may be composedof more than one optical element. In particular, a plurality of lensesmay be combined so as to form the imaging arrangement 902 or thefocusing arrangement 905. Likewise the dichotic filters may be composedof more than one filter or optic element. In particular to composeparticular properties of the respective arrangements. The focusingoptical arrangement and the semiconductor photo sensor may be arrangedto each other that the beam of light is focused to a spot having adiameter of less than 1.0 mm, particularly of less than 0.6 mm. Inparticular when using semiconductor sensors the signal to noise rationSNR can be significantly reduced. As can be seen in FIG. 25, thewavelength division multiplexer as described above may further comprisean image relay optical arrangement 907. This image relay opticalarrangement may be located in or in proximity to an image plane producedby said imaging optical arrangement in the second branch, wherein saidimage relay optical arrangement is adapted to produce an image of saidimaging optical arrangement in a third branch having substantially thesame size as the image in the second branch. The third branch in FIG. 25is the beam traveling from the element 907 toward element 909. Theeffective size of the optical element is the area where beams from theobject transit the optical element. Consequently, producing an image ofsubstantially the same size as the effective size of said opticalelement means that between the optical element and the image the beam iswithin a virtual parallel tube. For illustration purposes, in thehypothetical case the object is a pinhole the optical element produces acollimated beam. The image relay optical arrangement 907 may be aconcave mirror. Alternatively the image relay optical arrangement 907may be a combination of a lens and a mirror, in particular a planemirror. The wavelength division multiplexer as described above mayfurther comprise an additional dichroic filter arrangement 909, whereinthe additional dichroic filter arrangement is located between said imagerelay optical arrangement 907 and an image produced by said image relayoptical arrangement 907. Said additional dichroic filter arrangement 909from the third branch produces a fourth branch and a fifth branch of thebeam of light having distinctive colors. The fourth branch in FIG. 25 isthe beam traveling from the dichroic filter 909 toward the focusingelement 908, whereas the fifth branch is the beam traveling from thedichroic filter toward the element 910. The above described wavelengthdivision multiplexer further may comprise an additional focusing opticalarrangement 908 and an additional semiconductor photo detector, whereinthe additional focusing optical arrangement 908 is located in the fourthbranch and focuses the beam of light in the fourth branch so as to focusthe beam of light onto the additional semiconductor photo detector. Thewavelength division multiplexer may further comprise a plurality ofimage relay optical arrangements 910, 911, 912, 913, a plurality ofdichroic filter arrangements 914, 915, 916, 917, a plurality ofsemiconductor photo detectors and a plurality of focusing opticalarrangements 918, 919, 920, 921, wherein each of the plurality offocusing optical arrangements is arranged between a respective one ofthe plurality of dichroic filter arrangements and a respective one ofthe plurality of semiconductor photo detectors so as to form a cascadedarrangement, as can be seen in FIG. 25. The additional focused spots mayhave a diameter of less than 1.0 mm, particularly of less than 0.6 mmfor multiple colored bands of said beam of light. The plurality ofdichroic filter arrangements are arranged in a common plane, as can beseen in FIG. 25A. The wavelength division multiplexer may furthercomprise a plan-parallel optical basis having a first surface and secondsurface parallel thereto, wherein the plurality of dichroic filterarrangements are arranged parallel, preferably in abutment to the firstsurface of the plan-parallel optical basis, as described with respect tothe WDM 90. The dichroic filter arrangements are assembled using atemplate that includes two optically flat glass plates bonded togetherin optical contact, wherein the dichroic filter arrangements are bondedto a filter holder using the template such that a coated filter surfaceof the dichroic filter arrangements are indented and optically parallelto a reference surface of the filter holder, as can be seen in FIGS.29A, B and C. The reference surface of the filter holder rests againstan optically flat surface of an reference block included in thewavelength division multiplexer thereby providing consistent opticalalignment when installing the dichroic filter arrangements into thewavelength division multiplexer. The respective image relay opticalarrangements may be formed into the second surface of the plan-paralleloptical basis, as can be seen in FIG. 25A. At least one of thesemiconductor photo detectors is an avalanche photo diode detector. Asan alternative or in addition, at least one of the semiconductor photodetectors is a carbon nanotube detector.

INDUSTRIAL APPLICABILITY

Although an embodiment of the present disclosure of an LD based opticalsystem for flow cytometric application has been described in somedetail, and equally advantageous embodiments have also been describedfor a stream based flow cytometric instrument, it will be apparent tothose of ordinary skill in the art that many modifications andvariations of the described embodiment are possible in the light of theabove teachings without departing from the principles and concepts ofthe disclosure as set forth in the claims.

Although an embodiment of the present disclosure of wavelength divisionmultiplexing device for separating light beam from an extended lightsource into multiple color bands has been described in some detail, andseveral other equally advantageous embodiments have also been described,it will be apparent to those ordinary skilled in the art that manymodifications and variations of the described embodiments are possiblein the light of the above teachings without departing from theprinciples and concepts of the disclosure as set forth in the claims.

Although the present disclosure describes certain exemplary embodiments,it is to be understood that such disclosure is purely illustrative andis not to be interpreted as limiting. Consequently, without departingfrom the spirit and scope of the disclosure, various alterations,modifications, and/or alternative applications of the disclosure will,no doubt, be suggested to those skilled in the art after having read thepreceding disclosure. Accordingly, it is intended that the followingclaims be interpreted as encompassing all alterations, modifications, oralternative applications as fall within the true spirit and scope of thedisclosure.

1.-363. (canceled)
 364. A flow cytometer having a laser diode (LD)system, the LD system comprising: an LD for emitting a diverging beam oflight from an edge thereof, the diverging beam of light having anelliptically-shaped cross-sectional profile with both a major axis and aminor axis; a collimating lens configured to convert the diverging beamof light emitted from the LD into a collimated elliptical beam of light,wherein the minor axis of the collimated elliptical beam of light isoriented parallel to a direction in which particles pass through aviewing zone; a beam compressing optical element configured to reduce asize of the collimated elliptical beam of light at the viewing zonewhereby a width of the collimated elliptical beam of light orientedperpendicular to the direction in which the particles pass through theviewing zone is less than a width of a liquid sheath flow; and acylindrical focusing element positioned adjacent to the viewing zonewith an axis of the cylindrical focusing element being orientedperpendicular to the direction in which the particles pass through theviewing zone whereby the minor axis of the elliptical beam of lightbecomes focused at the viewing zone and the size of the major axis ofthe elliptical beam of light at the viewing zone remains essentiallyunchanged.
 365. The flow cytometer of claim 364, wherein the beamcompressing optical element is configured to reduce the size of both themajor axis and the minor axis of the collimated elliptical beam oflight.
 366. The flow cytometer of claim 364, wherein the beamcompressing optical element is a spherical focusing lens.
 367. The flowcytometer of claim 364, wherein the beam compressing optical element isa plano-convex lens.
 368. The flow cytometer of claim 364, wherein thebeam compressing optical element is an achromatic doublet lens.
 369. Theflow cytometer of claim 364, wherein the beam compressing opticalelement is one of a combination of spherical lenses, cylindrical lenses,and/or prism pairs.
 370. The flow cytometer of claim 364, wherein thebeam compressing optical element is a concave mirror.
 371. The flowcytometer of claim 364, wherein the LD system further comprises apolarization conditioning element disposed between the collimating lensand the beam compressing optical element through which the collimatedelliptical beam of light passes.
 372. The flow cytometer of claim 364,wherein the cylindrical focusing element is a cylindrical plano-convexlens.
 373. The flow cytometer of claim 364 further having a cuvette,wherein the viewing zone is located within a channel located within thecuvette.
 374. The flow cytometer of claim 373, wherein the cylindricalfocusing element is attached to the cuvette.
 375. The flow cytometer ofclaim 373, wherein the cylindrical focusing element is optically coupledto an abutting flat surface of the cuvette.
 376. The flow cytometer ofclaim 364, wherein the viewing zone is located in a jet stream formed bysample liquid and the liquid sheath flow.
 377. A flow cytometer having alaser diode (LD) system, the LD system comprising: an LD for emitting adiverging beam of light from an edge thereof, the diverging beam oflight having an elliptically-shaped cross-sectional profile with both afast axis and a slow axis; a collimating lens configured to convert thediverging beam of light emitted from the LD into a collimated ellipticalbeam of light, wherein the slow axis of the collimated elliptical beamof light is oriented parallel to a direction in which particles passthrough a viewing zone; a beam compressing optical element configured toreduce the size of both the fast axis and the slow axis of theelliptical beam of light; and a cylindrical focusing element positionedadjacent to the viewing zone with an axis of the cylindrical focusingelement being oriented perpendicular to the direction in which theparticles pass through the viewing zone whereby the slow axis of theelliptical beam of light becomes focused at the viewing zone and thesize of the fast axis of the elliptical beam of light at the viewingzone remains essentially unchanged.
 378. The flow cytometer of claim377, wherein the beam compressing optical element is configured to focusthe fast axis of the elliptical beam of light at the viewing zone. 379.The flow cytometer of claim 378, wherein the fast axis of the ellipticalbeam of light is focused at the viewing zone to a size less than a widthof a liquid sheath flow.
 380. The flow cytometer of claim 379, whereinthe cylindrical focusing element is optically coupled to a cuvette,wherein the viewing zone is located within a channel located within thecuvette.
 381. The flow cytometer of claim 377, wherein the LD systemfurther comprises a reflecting optical element configured to orient theslow axis of the collimated elliptical beam of light parallel to thedirection in which particles pass through the viewing zone.
 382. Amethod for delivering an elliptically shaped beam of light using a laserdiode (LD) system, the elliptically shaped beam of light having a smoothprofile at a focus of a minor axis thereof that is located at a viewingzone through which a sample liquid flows, the sample liquid beinghydrodynamically focused within the viewing zone by a liquid sheath flowthat also flows through the viewing zone, the method comprising thesteps of: providing an LD that emits a diverging beam of light from anedge thereof, the diverging beam of light having an elliptically shapedcross-sectional profile with both a major axis and a minor axis;impinging the diverging beam of light emitted by the LD upon acollimating lens to convert the diverging beam of light emittedtherefrom into a collimated elliptical beam of light wherein the minoraxis of said collimated elliptical beam of light is oriented parallel toa direction in which sample liquid passes through the viewing zone;after passing through the collimating lens, impinging the collimatedelliptical beam of light upon a beam compressing optical element toreduce a size of the collimated elliptical beam of light at the viewingzone whereby a width of the major axis of the collimated elliptical beamof light oriented perpendicular to the direction in which sample liquidpasses through the viewing zone becomes less than a width of said liquidsheath flow; and after passing through the beam compressing opticalelement, impinging the elliptical beam of light upon a cylindricalfocusing element positioned adjacent to the viewing zone with an axis ofthe cylindrical focusing element being oriented perpendicular to thedirection in which sample liquid passes through the viewing zone,whereby the minor axis of the elliptical beam of light becomes focusedat the viewing zone and the size of the major axis of the ellipticalbeam of light at the viewing zone remains essentially unchanged. 383.The method of claim 382, wherein the viewing zone is in a cuvette,wherein the beam compressing optical element is attached to the cuvette.